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Sample records for mycobacterium tuberculosis proteasome

  1. Identification of substrates of the Mycobacterium tuberculosis proteasome

    PubMed Central

    Pearce, Michael J; Arora, Pooja; Festa, Richard A; Butler-Wu, Susan M; Gokhale, Rajesh S; Darwin, K Heran

    2006-01-01

    The putative proteasome-associated proteins Mpa (Mycobaterium proteasomal ATPase) and PafA (proteasome accessory factor A) of the human pathogen Mycobacterium tuberculosis (Mtb) are essential for virulence and resistance to nitric oxide. However, a direct link between the proteasome protease and Mpa or PafA has never been demonstrated. Furthermore, protein degradation by bacterial proteasomes in vitro has not been accomplished, possibly due to the failure to find natural degradation substrates or other necessary proteasome co-factors. In this work, we identify the first bacterial proteasome substrates, malonyl Co-A acyl carrier protein transacylase and ketopantoate hydroxymethyltransferase, enzymes that are required for the biosynthesis of fatty acids and polyketides that are essential for the pathogenesis of Mtb. Maintenance of the physiological levels of these enzymes required Mpa and PafA in addition to proteasome protease activity. Mpa levels were also regulated in a proteasome-dependent manner. Finally, we found that a conserved tyrosine of Mpa was essential for function. Thus, these results suggest that Mpa, PafA, and the Mtb proteasome degrade bacterial proteins that are important for virulence in mice. PMID:17082771

  2. Structural Insights on the Mycobacterium tuberculosis Proteasomal ATPase Mpa

    SciTech Connect

    Wang, T.; Li, H; Lin, G; Tang, C; Li, D; Nathan, C; Heran Darwin, K

    2009-01-01

    Proteasome-mediated protein turnover in all domains of life is an energy-dependent process that requires ATPase activity. Mycobacterium tuberculosis (Mtb) was recently shown to possess a ubiquitin-like proteasome pathway that plays an essential role in Mtb resistance to killing by products of host macrophages. Here we report our structural and biochemical investigation of Mpa, the presumptive Mtb proteasomal ATPase. We demonstrate that Mpa binds to the Mtb proteasome in the presence of ATPS, providing the physical evidence that Mpa is the proteasomal ATPase. X-ray crystallographic determination of the conserved interdomain showed a five stranded double {beta} barrel structure containing a Greek key motif. Structure and mutational analysis indicate a major role of the interdomain for Mpa hexamerization. Our mutational and functional studies further suggest that the central channel in the Mpa hexamer is involved in protein substrate translocation and degradation. These studies provide insights into how a bacterial proteasomal ATPase interacts with and facilitates protein degradation by the proteasome.

  3. Proteasomal control of cytokinin synthesis protects Mycobacterium tuberculosis against nitric oxide

    PubMed Central

    Samanovic, Marie I.; Tu, Shengjiang; Novák, Ondřej; Iyer, Lakshminarayan M.; McAllister, Fiona E.; Aravind, L.; Gygi, Steven P.; Hubbard, Stevan R.; Strnad, Miroslav; Darwin, K. Heran

    2015-01-01

    Summary One of several roles of the Mycobacterium tuberculosis proteasome is to defend against host-produced nitric oxide (NO), a free radical that can damage numerous biological macromolecules. Mutations that inactivate proteasomal degradation in Mycobacterium tuberculosis result in bacteria that are hypersensitive to NO and attenuated for growth in vivo, but it was not known why. To elucidate the link between proteasome function, NO-resistance, and pathogenesis, we screened for suppressors of NO hypersensitivity in a mycobacterial proteasome ATPase mutant and identified mutations in Rv1205. We determined that Rv1205 encodes a pupylated proteasome substrate. Rv1205 is a homologue of the plant enzyme LONELY GUY, which catalyzes the production of hormones called cytokinins. Remarkably, we report for the first time that an obligate human pathogen secretes several cytokinins. Finally, we determined that the Rv1205-dependent accumulation of cytokinin breakdown products is likely responsible for the sensitization of Mycobacterium tuberculosis proteasome-associated mutants to NO. PMID:25728768

  4. Structural Basis for the Assembly and Gate Closure Mechanisms of the Mycobacterium tuberculosis 20S Proteasome

    SciTech Connect

    Lin, D.; Li, H; Wang, T; Pan, H; Lin, G; Li, H

    2010-01-01

    Mycobacterium tuberculosis (Mtb) possesses a proteasome system analogous to the eukaryotic ubiquitin-proteasome pathway. Mtb requires the proteasome to resist killing by the host immune system. The detailed assembly process and the gating mechanism of Mtb proteasome have remained unknown. Using cryo-electron microscopy and X-ray crystallography, we have obtained structures of three Mtb proteasome assembly intermediates, showing conformational changes during assembly, and explaining why the {beta}-subunit propeptide inhibits rather than promotes assembly. Although the eukaryotic proteasome core particles close their protein substrate entrance gates with different amino terminal peptides of the seven {alpha}-subunits, it has been unknown how a prokaryotic proteasome might close the gate at the symmetry axis with seven identical peptides. We found in the new Mtb proteasome crystal structure that the gate is tightly sealed by the seven identical peptides taking on three distinct conformations. Our work provides the structural bases for assembly and gating mechanisms of the Mtb proteasome.

  5. Structural basis for the assembly and gate closure mechanisms of the Mycobacterium tuberculosis 20S proteasome

    SciTech Connect

    Li, D.; Li, H.; Li, H.; Wang, T.; Pan, H.; Lin, G.

    2010-06-16

    Mycobacterium tuberculosis (Mtb) possesses a proteasome system analogous to the eukaryotic ubiquitin-proteasome pathway. Mtb requires the proteasome to resist killing by the host immune system. The detailed assembly process and the gating mechanism of Mtb proteasome have remained unknown. Using cryo-electron microscopy and X-ray crystallography, we have obtained structures of three Mtb proteasome assembly intermediates, showing conformational changes during assembly, and explaining why the {beta}-subunit propeptide inhibits rather than promotes assembly. Although the eukaryotic proteasome core particles close their protein substrate entrance gates with different amino terminal peptides of the seven {alpha}-subunits, it has been unknown how a prokaryotic proteasome might close the gate at the symmetry axis with seven identical peptides. We found in the new Mtb proteasome crystal structure that the gate is tightly sealed by the seven identical peptides taking on three distinct conformations. Our work provides the structural bases for assembly and gating mechanisms of the Mtb proteasome.

  6. An adenosine triphosphate-independent proteasome activator contributes to the virulence of Mycobacterium tuberculosis

    DOE PAGESBeta

    Jastrab, Jordan B.; Wang, Tong; Murphy, J. Patrick; Bai, Lin; Hu, Kuan; Merkx, Remco; Huang, Jessica; Chatterjee, Champak; Ovaa, Huib; Gygi, Steven P.; et al

    2015-03-23

    Mycobacterium tuberculosis encodes a proteasome that is highly similar to eukaryotic proteasomes and is required to cause lethal infections in animals. The only pathway known to target proteins for proteasomal degradation in bacteria is pupylation, which is functionally analogous to eukaryotic ubiquitylation. However, evidence suggests that the M. tuberculosis proteasome contributes to pupylation-independent pathways as well. To identify new proteasome cofactors that might contribute to such pathways, we isolated proteins that bound to proteasomes overproduced in M. tuberculosis and found a previously uncharacterized protein, Rv3780, which formed rings and capped M. tuberculosis proteasome core particles. Rv3780 enhanced peptide and proteinmore » degradation by proteasomes in an adenosine triphosphate (ATP)-independent manner. We identified putative Rv3780-dependent proteasome substrates and found that Rv3780 promoted robust degradation of the heat shock protein repressor, HspR. Importantly, an M. tuberculosis Rv3780 mutant had a general growth defect, was sensitive to heat stress, and was attenuated for growth in mice. Collectively, these data demonstrate that ATP-independent proteasome activators are not confined to eukaryotes and can contribute to the virulence of one the world’s most devastating pathogens.« less

  7. An adenosine triphosphate-independent proteasome activator contributes to the virulence of Mycobacterium tuberculosis

    SciTech Connect

    Jastrab, Jordan B.; Wang, Tong; Murphy, J. Patrick; Bai, Lin; Hu, Kuan; Merkx, Remco; Huang, Jessica; Chatterjee, Champak; Ovaa, Huib; Gygi, Steven P.; Li, Huilin; Darwin, K. Heran

    2015-03-23

    Mycobacterium tuberculosis encodes a proteasome that is highly similar to eukaryotic proteasomes and is required to cause lethal infections in animals. The only pathway known to target proteins for proteasomal degradation in bacteria is pupylation, which is functionally analogous to eukaryotic ubiquitylation. However, evidence suggests that the M. tuberculosis proteasome contributes to pupylation-independent pathways as well. To identify new proteasome cofactors that might contribute to such pathways, we isolated proteins that bound to proteasomes overproduced in M. tuberculosis and found a previously uncharacterized protein, Rv3780, which formed rings and capped M. tuberculosis proteasome core particles. Rv3780 enhanced peptide and protein degradation by proteasomes in an adenosine triphosphate (ATP)-independent manner. We identified putative Rv3780-dependent proteasome substrates and found that Rv3780 promoted robust degradation of the heat shock protein repressor, HspR. Importantly, an M. tuberculosis Rv3780 mutant had a general growth defect, was sensitive to heat stress, and was attenuated for growth in mice. Collectively, these data demonstrate that ATP-independent proteasome activators are not confined to eukaryotes and can contribute to the virulence of one the world’s most devastating pathogens.

  8. Fellutamide B is a Potent Inhibitor of the Mycobacterium tuberculosis Proteasome

    SciTech Connect

    Lin, G.; Li, D; Chidawanyika, T; Nathan, C; Li, H

    2010-01-01

    Via high-throughput screening of a natural compound library, we have identified a lipopeptide aldehyde, fellutamide B (1), as the most potent inhibitor of the Mycobacterium tuberculosis (Mtb) proteasome tested to date. Kinetic studies reveal that 1 inhibits both Mtb and human proteasomes in a time-dependent manner under steady-state condition. Remarkably, 1 inhibits the Mtb proteasome in a single-step binding mechanism with K{sub i} = 6.8 nM, whereas it inhibits the human proteasome {beta}5 active site following a two-step mechanism with K{sub i} = 11.5 nM and K*{sub i} = 0.93 nM. Co-crystallization of 1 bound to the Mtb proteasome revealed a structural basis for the tight binding of 1 to the active sites of the Mtb proteasome. The hemiacetal group of 1 in the Mtb proteasome takes the (R)-configuration, whereas in the yeast proteasome it takes the (S)-configuration, indicating that the pre-chiral CHO group of 1 binds to the active site Thr1 in a different orientation. Re-examination of the structure of the yeast proteasome in complex with 1 showed significant conformational changes at the substrate-binding cleft along the active site. These structural differences are consistent with the different kinetic mechanisms of 1 against Mtb and human proteasomes.

  9. Structure of the Mycobacterium tuberculosis proteasome and mechanism of inhibition by a peptidyl boronate

    SciTech Connect

    Hu,G.; Lin, G.; Wang, M.; Dick, L.; Xu, R.; Nathan, C.; Li, H.

    2006-01-01

    Mycobacterium tuberculosis (Mtb) has the remarkable ability to resist killing by human macrophages. The 750 kDa proteasome, not available in most eubacteria except Actinomycetes, appears to contribute to Mtb's resistance. The crystal structure of the Mtb proteasome at 3.0 Angstroms resolution reveals a substrate-binding pocket with composite features of the distinct {beta}1, {beta}2 and {beta}5 substrate binding sites of eukaryotic proteasomes, accounting for the broad specificity of the Mtb proteasome towards oligopeptides described in the companion article [Lin et al. (2006), Mol Microbiol doi:10.1111/j.1365-2958.2005.05035.x]. The substrate entrance at the end of the cylindrical proteasome appears open in the crystal structure due to partial disorder of the a-subunit N-terminal residues. However, cryo-electron microscopy of the core particle reveals a closed end, compatible with the density observed in negative-staining electron microscopy that depended on the presence of the N-terminal octapeptides of the a-subunits in the companion article, suggesting that the Mtb proteasome has a gated structure. We determine for the first time the proteasomal inhibition mechanism of the dipeptidyl boronate N-(4-morpholine)carbonyl-{beta}-(1-naphthyl)-l-alanine-l-leucine boronic acid (MLN-273), an analogue of the antimyeloma drug bortezomib. The structure improves prospects for designing Mtb-specific proteasomal inhibitors as a novel approach to chemotherapy of tuberculosis.

  10. Structure of the Mycobacterium tuberculosis proteasome and mechanism of inhibition by a peptidyl boronate.

    PubMed

    Hu, Guiqing; Lin, Gang; Wang, Ming; Dick, Lawrence; Xu, Rui-Ming; Nathan, Carl; Li, Huilin

    2006-03-01

    Mycobacterium tuberculosis (Mtb) has the remarkable ability to resist killing by human macrophages. The 750 kDa proteasome, not available in most eubacteria except Actinomycetes, appears to contribute to Mtb's resistance. The crystal structure of the Mtb proteasome at 3.0 A resolution reveals a substrate-binding pocket with composite features of the distinct beta1, beta2 and beta5 substrate binding sites of eukaryotic proteasomes, accounting for the broad specificity of the Mtb proteasome towards oligopeptides described in the companion article [Lin et al. (2006), Mol Microbiol doi:10.1111/j.1365-2958.2005.05035.x]. The substrate entrance at the end of the cylindrical proteasome appears open in the crystal structure due to partial disorder of the alpha-subunit N-terminal residues. However, cryo-electron microscopy of the core particle reveals a closed end, compatible with the density observed in negative-staining electron microscopy that depended on the presence of the N-terminal octapetides of the alpha-subunits in the companion article, suggesting that the Mtb proteasome has a gated structure. We determine for the first time the proteasomal inhibition mechanism of the dipeptidyl boronate N-(4-morpholine)carbonyl-beta-(1-naphthyl)-L-alanine-L-leucine boronic acid (MLN-273), an analogue of the antimyeloma drug bortezomib. The structure improves prospects for designing Mtb-specific proteasomal inhibitors as a novel approach to chemotherapy of tuberculosis. PMID:16468986

  11. Structural analysis of the dodecameric proteasome activator PafE in Mycobacterium tuberculosis.

    PubMed

    Bai, Lin; Hu, Kuan; Wang, Tong; Jastrab, Jordan B; Darwin, K Heran; Li, Huilin

    2016-04-01

    The human pathogen Mycobacterium tuberculosis (Mtb) requires a proteasome system to cause lethal infections in mice. We recently found that proteasome accessory factor E (PafE, Rv3780) activates proteolysis by the Mtb proteasome independently of adenosine triphosphate (ATP). Moreover, PafE contributes to the heat-shock response and virulence of Mtb Here, we show that PafE subunits formed four-helix bundles similar to those of the eukaryotic ATP-independent proteasome activator subunits of PA26 and PA28. However, unlike any other known proteasome activator, PafE formed dodecamers with 12-fold symmetry, which required a glycine-XXX-glycine-XXX-glycine motif that is not found in previously described activators. Intriguingly, the truncation of the PafE carboxyl-terminus resulted in the robust binding of PafE rings to native proteasome core particles and substantially increased proteasomal activity, suggesting that the extended carboxyl-terminus of this cofactor confers suboptimal binding to the proteasome core particle. Collectively, our data show that proteasomal activation is not limited to hexameric ATPases in bacteria. PMID:27001842

  12. Structural analysis of the dodecameric proteasome activator PafE in Mycobacterium tuberculosis

    DOE PAGESBeta

    Bai, Lin; Hu, Kuan; Wang, Tong; Jastrab, Jordan B.; Darwin, K. Heran; Li, Huilin

    2016-03-21

    Here, the human pathogen Mycobacterium tuberculosis (Mtb) requires a proteasome system to cause lethal infections in mice. We recently found that proteasome accessory factor E (PafE, Rv3780) activates proteolysis by the Mtb proteasome independently of adenosine triphosphate (ATP). Moreover, PafE contributes to the heat-shock response and virulence of Mtb. Here, we show that PafE subunits formed four-helix bundles similar to those of the eukaryotic ATP-independent proteasome activator subunits of PA26 and PA28. However, unlike any other known proteasome activator, PafE formed dodecamers with 12-fold symmetry, which required a glycine-XXX-glycine-XXX-glycine motif that is not found in previously described activators. Intriguingly, themore » truncation of the PafE carboxyl-terminus resulted in the robust binding of PafE rings to native proteasome core particles and substantially increased proteasomal activity, suggesting that the extended carboxyl-terminus of this cofactor confers suboptimal binding to the proteasome core particle. Collectively, our data show that proteasomal activation is not limited to hexameric ATPases in bacteria.« less

  13. Distinct specificities of Mycobacterium tuberculosis and mammalian proteasomes for N-acetyl tripeptide substrates.

    PubMed

    Lin, Gang; Tsu, Christopher; Dick, Lawrence; Zhou, Xi K; Nathan, Carl

    2008-12-01

    The proteasome of Mycobacterium tuberculosis (Mtb) is a validated and drug-treatable target for therapeutics. To lay ground-work for developing peptide-based inhibitors with a useful degree of selectivity for the Mtb proteasome over those of the host, we used a library of 5,920 N-acetyl tripeptide-aminomethylcoumarins to contrast the substrate preferences of the recombinant Mtb proteasome wild type and open gate mutant, the Rhodococcus erythropolis proteasome, and the bovine proteasome with activator PA28. The Mtb proteasome was distinctive in strictly preferring P1 = tryptophan, particularly in combination with P3 = glycine, proline, lysine or arginine. Screening results were validated with Michalis-Menten kinetic analyses of 21 oligopeptide aminomethyl-coumarin substrates. Bortezomib, a proteasome inhibitor in clinical use, and 17 analogs varying only at P1 were used to examine the differential impact of inhibitors on human and Mtb proteasomes. The results with the inhibitor panel confirmed those with the substrate panel in demonstrating differential preferences of Mtb and mammalian proteasomes at the P1 amino acid. Changing P1 in bortezomib from Leu to m-CF(3)-Phe led to a 220-fold increase in IC(50) against the human proteasome, whereas changing a P1 Ala to m-F-Phe decreased the IC(50) 400-fold against the Mtb proteasome. The change of a P1 Ala to m-Cl-Phe led to an 8000-fold shift in inhibitory potency in favor of the Mtb proteasome, resulting in 8-fold selectivity. Combinations of preferred amino acids at different sites may thus improve the species selectivity of peptide-based inhibitors that target the Mtb proteasome. PMID:18829465

  14. The Mycobacterium tuberculosis Proteasome Active Site Threonine Is Essential for Persistence Yet Dispensable for Replication and Resistance to Nitric Oxide

    PubMed Central

    Gandotra, Sheetal; Lebron, Maria B.; Ehrt, Sabine

    2010-01-01

    Previous work revealed that conditional depletion of the core proteasome subunits PrcB and PrcA impaired growth of Mycobacterium tuberculosis in vitro and in mouse lungs, caused hypersusceptibility to nitric oxide (NO) and impaired persistence of the bacilli during chronic mouse infections. Here, we show that genetic deletion of prcBA led to similar phenotypes. Surprisingly, however, an active site mutant proteasome complemented the in vitro and in vivo growth defects of the prcBA knockout (ΔprcBA) as well as its NO hypersensitivity. In contrast, long-term survival of M. tuberculosis in stationary phase and during starvation in vitro and in the chronic phase of mouse infection required a proteolytically active proteasome. Inhibition of inducible nitric oxide synthase did not rescue survival of ΔprcBA, revealing a function beyond NO defense, by which the proteasome contributes to M. tuberculosis fitness during chronic mouse infections. These findings suggest that proteasomal proteolysis facilitates mycobacterial persistence, that M. tuberculosis faces starvation during chronic mouse infections and that the proteasome serves a proteolysis-independent function. PMID:20711362

  15. Optimization and Evaluation of 5-Styryl-Oxathiazol-2-one Mycobacterium tuberculosis Proteasome Inhibitors as Potential Antitubercular Agents

    PubMed Central

    Russo, Francesco; Gising, Johan; Åkerbladh, Linda; Roos, Annette K; Naworyta, Agata; Mowbray, Sherry L; Sokolowski, Anders; Henderson, Ian; Alling, Torey; Bailey, Mai A; Files, Megan; Parish, Tanya; Karlén, Anders; Larhed, Mats

    2015-01-01

    This is the first report of 5-styryl-oxathiazol-2-ones as inhibitors of the Mycobacterium tuberculosis (Mtb) proteasome. As part of the study, the structure–activity relationship of oxathiazolones as Mtb proteasome inhibitors has been investigated. Furthermore, the prepared compounds displayed a good selectivity profile for Mtb compared to the human proteasome. The 5-styryl-oxathiazol-2-one inhibitors identified showed little activity against replicating Mtb, but were rapidly bactericidal against nonreplicating bacteria. (E)-5-(4-Chlorostyryl)-1,3,4-oxathiazol-2-one) was most effective, reducing the colony-forming units (CFU)/mL below the detection limit in only seven days at all concentrations tested. The results suggest that this new class of Mtb proteasome inhibitors has the potential to be further developed into novel antitubercular agents for synergistic combination therapies with existing drugs. PMID:26246997

  16. Binding-induced folding of prokaryotic ubiquitin-like protein on the mycobacterium proteasomal ATPase targets substrates for degradation

    SciTech Connect

    Wang, T.; Li, H.; Darwin, K. H.

    2010-11-01

    Mycobacterium tuberculosis uses a proteasome system that is analogous to the eukaryotic ubiquitin-proteasome pathway and is required for pathogenesis. However, the bacterial analog of ubiquitin, prokaryotic ubiquitin-like protein (Pup), is an intrinsically disordered protein that bears little sequence or structural resemblance to the highly structured ubiquitin. Thus, it was unknown how pupylated proteins were recruited to the proteasome. Here, we show that the Mycobacterium proteasomal ATPase (Mpa) has three pairs of tentacle-like coiled coils that recognize Pup. Mpa bound unstructured Pup through hydrophobic interactions and a network of hydrogen bonds, leading to the formation of an {alpha}-helix in Pup. Our work describes a binding-induced folding recognition mechanism in the Pup-proteasome system that differs mechanistically from substrate recognition in the ubiquitin-proteasome system. This key difference between the prokaryotic and eukaryotic systems could be exploited for the development of a small molecule-based treatment for tuberculosis.

  17. Binding-induced Folding of Prokaryotic Ubiquitin-like Protein on the Mycobacterium Proteasomal ATPase Targets Substrates for Degradation

    SciTech Connect

    T Wang; K Heran Darwin; H Li

    2011-12-31

    Mycobacterium tuberculosis uses a proteasome system that is analogous to the eukaryotic ubiquitin-proteasome pathway and is required for pathogenesis. However, the bacterial analog of ubiquitin, prokaryotic ubiquitin-like protein (Pup), is an intrinsically disordered protein that bears little sequence or structural resemblance to the highly structured ubiquitin. Thus, it was unknown how pupylated proteins were recruited to the proteasome. Here, we show that the Mycobacterium proteasomal ATPase (Mpa) has three pairs of tentacle-like coiled coils that recognize Pup. Mpa bound unstructured Pup through hydrophobic interactions and a network of hydrogen bonds, leading to the formation of an {alpha}-helix in Pup. Our work describes a binding-induced folding recognition mechanism in the Pup-proteasome system that differs mechanistically from substrate recognition in the ubiquitin-proteasome system. This key difference between the prokaryotic and eukaryotic systems could be exploited for the development of a small molecule-based treatment for tuberculosis.

  18. Immune Responses in Cattle Inoculated with Mycobacterium bovis, Mycobacterium tuberculosis, or Mycobacterium kansasii

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cattle were inoculated with Mycobacterium bovis, Mycobacterium tuberculosis, or Mycobacterium kansasii to compare antigen-specific immune responses to varied patterns of mycobacterial disease. Disease expression ranged from colonization with associated pathology (M. bovis), colonization without path...

  19. Bacterial Proteasomes

    PubMed Central

    Jastrab, Jordan B.; Darwin, K. Heran

    2015-01-01

    Interest in bacterial proteasomes was sparked by the discovery that proteasomal degradation is required for the pathogenesis of Mycobacterium tuberculosis, one of the world's deadliest pathogens. Although bacterial proteasomes are structurally similar to their eukaryotic and archaeal homologs, there are key differences in their mechanisms of assembly, activation, and substrate targeting for degradation. In this article, we compare and contrast bacterial proteasomes with their archaeal and eukaryotic counterparts, and we discuss recent advances in our understanding of how bacterial proteasomes function to influence microbial physiology. PMID:26488274

  20. Peritoneal tuberculosis due to Mycobacterium caprae

    PubMed Central

    Nebreda, T.; Álvarez-Prida, E.; Blanco, B.; Remacha, M.A.; Samper, S.; Jiménez, M.S.

    2016-01-01

    The incidence of tuberculosis in humans due to Mycobacterium caprae is very low and is almost confined to Europe. We report a case of a previously healthy 41-year-old Moroccan with a 6 month history of abdominal pain, weight loss, fatigue and diarrhea. A diagnosis of peritoneal tuberculosis due to M. caprae was made. PMID:27134824

  1. Mycobacterium tuberculosis and the host response

    PubMed Central

    Kaufmann, Stefan H.E.; Cole, Stewart T.; Mizrahi, Valerie; Rubin, Eric; Nathan, Carl

    2005-01-01

    Mycobacterium tuberculosis remains a leading cause of morbidity and mortality worldwide. Advances reported at a recent international meeting highlight insights and controversies in the genetics of M. tuberculosis and the infected host, the nature of protective immune responses, adaptation of the bacillus to host-imposed stresses, animal models, and new techniques. PMID:15939785

  2. Drug Resistance Mechanisms in Mycobacterium tuberculosis

    PubMed Central

    Palomino, Juan Carlos; Martin, Anandi

    2014-01-01

    Tuberculosis (TB) is a serious public health problem worldwide. Its situation is worsened by the presence of multidrug resistant (MDR) strains of Mycobacterium tuberculosis, the causative agent of the disease. In recent years, even more serious forms of drug resistance have been reported. A better knowledge of the mechanisms of drug resistance of M. tuberculosis and the relevant molecular mechanisms involved will improve the available techniques for rapid drug resistance detection and will help to explore new targets for drug activity and development. This review article discusses the mechanisms of action of anti-tuberculosis drugs and the molecular basis of drug resistance in M. tuberculosis. PMID:27025748

  3. Pathway Profiling in Mycobacterium tuberculosis

    PubMed Central

    Thomas, Suzanne T.; VanderVen, Brian C.; Sherman, David R.; Russell, David G.; Sampson, Nicole S.

    2011-01-01

    Mycobacterium tuberculosis, the bacterium that causes tuberculosis, imports and metabolizes host cholesterol during infection. This ability is important in the chronic phase of infection. Here we investigate the role of the intracellular growth operon (igr), which has previously been identified as having a cholesterol-sensitive phenotype in vitro and which is important for intracellular growth of the mycobacteria. We have employed isotopically labeled low density lipoproteins containing either [1,7,15,22,26-14C]cholesterol or [1,7,15,22,26-13C]cholesterol and high resolution LC/MS as tools to profile the cholesterol-derived metabolome of an igr operon-disrupted mutant (Δigr) of M. tuberculosis. A partially metabolized cholesterol species accumulated in the Δigr knock-out strain that was absent in the complemented and parental wild-type strains. Structural elucidation by multidimensional 1H and 13C NMR spectroscopy revealed the accumulated metabolite to be methyl 1β-(2′-propanoate)-3aα-H-4α-(3′-propanoic acid)-7aβ-methylhexahydro-5-indanone. Heterologously expressed and purified FadE28-FadE29, an acyl-CoA dehydrogenase encoded by the igr operon, catalyzes the dehydrogenation of 2′-propanoyl-CoA ester side chains in substrates with structures analogous to the characterized metabolite. Based on the structure of the isolated metabolite, enzyme activity, and bioinformatic annotations, we assign the primary function of the igr operon to be degradation of the 2′-propanoate side chain. Therefore, the igr operon is necessary to completely metabolize the side chain of cholesterol metabolites. PMID:22045806

  4. Dormancy models for Mycobacterium tuberculosis: A minireview.

    PubMed

    Alnimr, Amani M

    2015-01-01

    Dormancy models for Mycobacterium tuberculosis play important roles in understanding various aspects of tuberculosis pathogenesis and in the testing of novel therapeutic regimens. By simulating the latent tuberculosis infection, in which the bacteria exist in a non-replicative state, the models demonstrate reduced susceptibility to antimycobacterial agents. This minireview outlines the models available for simulating latent tuberculosis both in vitro and in several animal species. Additionally, this minireview discusses the advantages and disadvantages of these models for investigating the bacterial subpopulations and susceptibilities to sterilization by various antituberculosis drugs. PMID:26413043

  5. Dormancy models for Mycobacterium tuberculosis: A minireview

    PubMed Central

    Alnimr, Amani M.

    2015-01-01

    Dormancy models for Mycobacterium tuberculosis play important roles in understanding various aspects of tuberculosis pathogenesis and in the testing of novel therapeutic regimens. By simulating the latent tuberculosis infection, in which the bacteria exist in a non-replicative state, the models demonstrate reduced susceptibility to antimycobacterial agents. This minireview outlines the models available for simulating latent tuberculosis both in vitro and in several animal species. Additionally, this minireview discusses the advantages and disadvantages of these models for investigating the bacterial subpopulations and susceptibilities to sterilization by various antituberculosis drugs. PMID:26413043

  6. Macrophage infection models for Mycobacterium tuberculosis.

    PubMed

    Johnson, Benjamin K; Abramovitch, Robert B

    2015-01-01

    Mycobacterium tuberculosis colonizes, survives, and grows inside macrophages. In vitro macrophage infection models, using both primary macrophages and cell lines, enable the characterization of the pathogen response to macrophage immune pressure and intracellular environmental cues. We describe methods to propagate and infect primary murine bone marrow-derived macrophages and J774 and THP-1 macrophage-like cell lines. We also present methods on the characterization of M. tuberculosis intracellular survival and the preparation of infected macrophages for imaging. PMID:25779326

  7. Mycobacterium tuberculosis wears what it eats

    PubMed Central

    Russell, David G.; VanderVen, Brian C.; Lee, Wonsik; Abramovitch, Robert B.; Kim, Mijeong; Homolka, Susanne; Niemann, Stefan; Rohde, Kyle H.

    2010-01-01

    Mycobacterium tuberculosis remains one of the most pernicious of human pathogens. Current vaccines are ineffective and drugs, although efficacious, require prolonged treatment with constant medical oversight. Overcoming these problems requires a greater appreciation of M. tuberculosis in the context of its host. Upon infection of either macrophages in culture or animal models, the bacterium re-aligns its metabolism in response to the new environments it encounters. Understanding these environments, and the stresses that they place on M. tuberculosis, should provide insights invaluable for the development of new chemo- and immuno-therapeutic strategies. PMID:20638643

  8. Radioimmunoassay of tuberculoprotein derived from Mycobacterium tuberculosis.

    PubMed Central

    Straus, E; Wu, N

    1980-01-01

    A radioimmunoassay was developed for constituent of the purified-protein derivative obtained from cultures of Mycobacterium tuberculosis. Crossreacting immunoreactive material was detected in cultures of other mycobacterial species, but no immunoreactivity was present in cultures of various fungal and bacterial species. The development of specific radioimmunoassays for tuberculoproteins offers a new research and diagnostic approach. Images PMID:6933481

  9. The Mycobacterium tuberculosis Cytochrome P450 System

    PubMed Central

    Ouellet, Hugues; Johnston, Jonathan B.; Ortiz de Montellano, Paul R.

    2009-01-01

    Tuberculosis remains a leading cause of human mortality. The emergence of strains of Mycobacterium tuberculosis, the causative agent, that are resistant to the major frontline antitubercular drugs increases the urgency for the development of new therapeutic agents. Sequencing of the M. tuberculosis genome revealed the existence of twenty cytochrome P450 enzymes, some of which are potential candidates for drug targeting. The recent burst of studies reporting microarray-based gene essentiality and transcriptome analyses under in vitro, ex vivo and in vivo conditions highlight the importance of selected P450 isoforms for M. tuberculosis viability and pathogenicity. Current knowledge of the structural and biochemical properties of the M. tuberculosis P450 enzymes and their putative redox partners is reviewed, with an emphasis on findings related to their physiological function(s) as well as their potential as drug targets. PMID:19635450

  10. [Coinfection of Mycobacterium malmoense and Mycobacterium tuberculosis in a patient with acquired inmune deficiency syndrome].

    PubMed

    Mederos Cuervo, Lilian María; Reyes Pérez, Angélica; Valdes Alonso, Lidunka; Rodríguez Delgado, Francisco; Sardiñas Aragón, Misleydis; Martínez Romero, María Rosarys; Díaz Romero, Raúl

    2014-01-01

    A case is presented of coinfection with Mycobacterium malmoense and Mycobacterium tuberculosis in a Cuban patient with AIDS which produced respiratory and liver disease respectively. Cultures done from sputum samples showed the presence of a non-pigmented, slow growing mycobacterial strain belonging to Runyon group III and identified as Mycobacterium malmoense. From cultures of liver tissue removed laparoscopically, a strain was isolated and subsequently identified as Mycobacterium tuberculosis. Anatomapathologic examination confirmed the diagnosis of tuberculosis, the patient received specific treatment and had a favorable clinical course. This report of a rare case of coinfection of Mycobacterium describes the first report of hepatic tuberculosis in a patient with AIDS in Cuba. PMID:25597735

  11. Porins increase copper susceptibility of Mycobacterium tuberculosis.

    PubMed

    Speer, Alexander; Rowland, Jennifer L; Haeili, Mehri; Niederweis, Michael; Wolschendorf, Frank

    2013-11-01

    Copper resistance mechanisms are crucial for many pathogenic bacteria, including Mycobacterium tuberculosis, during infection because the innate immune system utilizes copper ions to kill bacterial intruders. Despite several studies detailing responses of mycobacteria to copper, the pathways by which copper ions cross the mycobacterial cell envelope are unknown. Deletion of porin genes in Mycobacterium smegmatis leads to a severe growth defect on trace copper medium but simultaneously increases tolerance for copper at elevated concentrations, indicating that porins mediate copper uptake across the outer membrane. Heterologous expression of the mycobacterial porin gene mspA reduced growth of M. tuberculosis in the presence of 2.5 μM copper by 40% and completely suppressed growth at 15 μM copper, while wild-type M. tuberculosis reached its normal cell density at that copper concentration. Moreover, the polyamine spermine, a known inhibitor of porin activity in Gram-negative bacteria, enhanced tolerance of M. tuberculosis for copper, suggesting that copper ions utilize endogenous outer membrane channel proteins of M. tuberculosis to gain access to interior cellular compartments. In summary, these findings highlight the outer membrane as the first barrier against copper ions and the role of porins in mediating copper uptake in M. smegmatis and M. tuberculosis. PMID:24013632

  12. Virulence factors of the Mycobacterium tuberculosis complex

    PubMed Central

    Forrellad, Marina A.; Klepp, Laura I.; Gioffré, Andrea; Sabio y García, Julia; Morbidoni, Hector R.; Santangelo, María de la Paz; Cataldi, Angel A.; Bigi, Fabiana

    2013-01-01

    The Mycobacterium tuberculosis complex (MTBC) consists of closely related species that cause tuberculosis in both humans and animals. This illness, still today, remains to be one of the leading causes of morbidity and mortality throughout the world. The mycobacteria enter the host by air, and, once in the lungs, are phagocytated by macrophages. This may lead to the rapid elimination of the bacillus or to the triggering of an active tuberculosis infection. A large number of different virulence factors have evolved in MTBC members as a response to the host immune reaction. The aim of this review is to describe the bacterial genes/proteins that are essential for the virulence of MTBC species, and that have been demonstrated in an in vivo model of infection. Knowledge of MTBC virulence factors is essential for the development of new vaccines and drugs to help manage the disease toward an increasingly more tuberculosis-free world. PMID:23076359

  13. Mycobacterium tuberculosis produces pili during human infection

    PubMed Central

    Alteri, Christopher J.; Xicohténcatl-Cortes, Juan; Hess, Sonja; Caballero-Olín, Guillermo; Girón, Jorge A.; Friedman, Richard L.

    2007-01-01

    Mycobacterium tuberculosis is responsible for nearly 3 million human deaths worldwide every year. Understanding the mechanisms and bacterial factors responsible for the ability of M. tuberculosis to cause disease in humans is critical for the development of improved treatment strategies. Many bacterial pathogens use pili as adherence factors to colonize the host. We discovered that M. tuberculosis produces fine (2- to 3-nm-wide), aggregative, flexible pili that are recognized by IgG antibodies contained in sera obtained from patients with active tuberculosis, indicating that the bacilli produce pili or pili-associated antigen during human infection. Purified M. tuberculosis pili (MTP) are composed of low-molecular-weight protein subunits encoded by the predicted M. tuberculosis H37Rv ORF, designated Rv3312A. MTP bind to the extracellular matrix protein laminin in vitro, suggesting that MTP possess adhesive properties. Isogenic mtp mutants lost the ability to produce Mtp in vitro and demonstrated decreased laminin-binding capabilities. MTP shares morphological, biochemical, and functional properties attributed to bacterial pili, especially with curli amyloid fibers. Thus, we propose that MTP are previously unidentified host-colonization factors of M. tuberculosis. PMID:17360408

  14. Mycobacterium tuberculosis Serine/Threonine Protein Kinases

    PubMed Central

    PRISIC, SLADJANA; HUSSON, ROBERT N.

    2014-01-01

    The Mycobacterium tuberculosis genome encodes 11 serine/threonine protein kinases (STPKs). A similar number of two-component systems are also present, indicating that these two signal transduction mechanisms are both important in the adaptation of this bacterial pathogen to its environment. The M. tuberculosis phosphoproteome includes hundreds of Ser- and Thr-phosphorylated proteins that participate in all aspects of M. tuberculosis biology, supporting a critical role for the STPKs in regulating M. tuberculosis physiology. Nine of the STPKs are receptor type kinases, with an extracytoplasmic sensor domain and an intracellular kinase domain, indicating that these kinases transduce external signals. Two other STPKs are cytoplasmic and have regulatory domains that sense changes within the cell. Structural analysis of some of the STPKs has led to advances in our understanding of the mechanisms by which these STPKs are activated and regulated. Functional analysis has provided insights into the effects of phosphorylation on the activity of several proteins, but for most phosphoproteins the role of phosphorylation in regulating function is unknown. Major future challenges include characterizing the functional effects of phosphorylation for this large number of phosphoproteins, identifying the cognate STPKs for these phosphoproteins, and determining the signals that the STPKs sense. Ultimately, combining these STPK-regulated processes into larger, integrated regulatory networks will provide deeper insight into M. tuberculosis adaptive mechanisms that contribute to tuberculosis pathogenesis. Finally, the STPKs offer attractive targets for inhibitor development that may lead to new therapies for drug-susceptible and drug-resistant tuberculosis. PMID:25429354

  15. Mycobacterium tuberculosis: Manipulator of Protective Immunity

    PubMed Central

    Korb, Vanessa C.; Chuturgoon, Anil A.; Moodley, Devapregasan

    2016-01-01

    Mycobacterium tuberculosis (MTB) is one of the most successful pathogens in human history and remains a global health challenge. MTB has evolved a plethora of strategies to evade the immune response sufficiently to survive within the macrophage in a bacterial-immunological equilibrium, yet causes sufficient immunopathology to facilitate its transmission. This review highlights MTB as the driver of disease pathogenesis and presents evidence of the mechanisms by which MTB manipulates the protective immune response into a pathological productive infection. PMID:26927066

  16. Comparative Mycobacterium tuberculosis spoligotype distribution in Mexico.

    PubMed

    Vera-Cabrera, Lucio; Ramos-Alvarez, Jessica; Molina-Torres, Carmen A; Rivera-Morales, Lydia Guadalupe; Rendón, Adrian; Quiñones-Falconi, Francisco; Ocampo-Candiani, Jorge

    2014-08-01

    In the present work, we studied the genetic diversity of Mycobacterium tuberculosis clinical isolates from patients according to their gender, age, and geographic location in Mexico. We did not observe any statistically significant differences in regard to age or gender. We found that spoligo international type 53 (SIT53) is more frequent in the northern states and that SIT119 predominates in central Mexico. PMID:24850349

  17. Comparative Mycobacterium tuberculosis Spoligotype Distribution in Mexico

    PubMed Central

    Ramos-Alvarez, Jessica; Molina-Torres, Carmen A.; Rivera-Morales, Lydia Guadalupe; Rendón, Adrian; Quiñones-Falconi, Francisco; Ocampo-Candiani, Jorge

    2014-01-01

    In the present work, we studied the genetic diversity of Mycobacterium tuberculosis clinical isolates from patients according to their gender, age, and geographic location in Mexico. We did not observe any statistically significant differences in regard to age or gender. We found that spoligo international type 53 (SIT53) is more frequent in the northern states and that SIT119 predominates in central Mexico. PMID:24850349

  18. Targeting the histidine pathway in Mycobacterium tuberculosis.

    PubMed

    Lunardi, Juleane; Nunes, José Eduardo S; Bizarro, Cristiano V; Basso, Luiz Augusto; Santos, Diógenes Santiago; Machado, Pablo

    2013-01-01

    Worldwide, tuberculosis is the leading cause of morbidity and mortality due to a single bacterial pathogen, Mycobacterium tuberculosis (Mtb). The increasing prevalence of this disease, the emergence of multi-, extensively, and totally drug-resistant strains, complicated by co-infection with the human immunodeficiency virus, and the length of tuberculosis chemotherapy have led to an urgent and continued need for the development of new and more effective antitubercular drugs. Within this context, the L-histidine biosynthetic pathway, which converts 5-phosphoribosyl 1-pyrophosphate to L-histidine in ten enzymatic steps, has been reported as a promising target of antimicrobial agents. This pathway is found in bacteria, archaebacteria, lower eukaryotes, and plants but is absent in mammals, making these enzymes highly attractive targets for the drug design of new antimycobacterial compounds with selective toxicity. Moreover, the biosynthesis of L-histidine has been described as essential for Mtb growth in vitro. Accordingly, a comprehensive overview of Mycobacterium tuberculosis histidine pathway enzymes as attractive targets for the development of new antimycobacterial agents is provided, mainly summarizing the previously reported inhibition data for Mtb or orthologous proteins. PMID:24111909

  19. Efficacies of selected disinfectants against Mycobacterium tuberculosis.

    PubMed

    Best, M; Sattar, S A; Springthorpe, V S; Kennedy, M E

    1990-10-01

    The activities of 10 formulations as mycobactericidal agents in Mycobacterium tuberculosis-contaminated suspensions (suspension test) and stainless steel surfaces (carrier test) were investigated with sputum as the organic load. The quaternary ammonium compound, chlorhexidine gluconate, and an iodophor were ineffective in all tests. Ethanol (70%) was effective against M. tuberculosis only in suspension in the absence of sputum. Povidone-iodine was not as efficacious when the test organism was dried on a surface as it was in suspension, and its activity was further reduced in the presence of sputum. Sodium hypochlorite required a higher concentration of available chlorine to achieve an effective level of disinfection than did sodium dichloroisocyanurate. Phenol (5%) was effective under all test conditions, producing at least a 4-log10 reduction in CFU. The undiluted glutaraldehyde-phenate solution was effective against M. tuberculosis and a second test organism, Mycobacterium smegmatis, even in the presence of dried sputum, whereas the diluted solution (1:16) was only effective against M. smegmatis in the suspension test. A solution of 2% glutaraldehyde was effective against M. tuberculosis. This investigation presents tuberculocidal efficacy data generated by methods simulating actual practices of routine disinfection. PMID:2121783

  20. Mycobacterium tuberculosis infection and vaccine development.

    PubMed

    Tang, Jiansong; Yam, Wing-Cheong; Chen, Zhiwei

    2016-05-01

    Following HIV/AIDS, tuberculosis (TB) continues to be the second most deadly infectious disease in humans. The global TB prevalence has become worse in recent years due to the emergence of multi-drug resistant (MDR) and extensively-drug resistant (XDR) strains, as well as co-infection with HIV. Although Bacillus Calmette-Guérin (BCG) vaccine has nearly been used for a century in many countries, it does not protect adult pulmonary tuberculosis and even causes disseminated BCG disease in HIV-positive population. It is impossible to use BCG to eliminate the Mycobacterium tuberculosis (M. tb) infection or to prevent TB onset and reactivation. Consequently, novel vaccines are urgently needed for TB prevention and immunotherapy. In this review, we discuss the TB prevalence, interaction between M. tb and host immune system, as well as recent progress of TB vaccine research and development. PMID:27156616

  1. High Persister Mutants in Mycobacterium tuberculosis.

    PubMed

    Torrey, Heather L; Keren, Iris; Via, Laura E; Lee, Jong Seok; Lewis, Kim

    2016-01-01

    Mycobacterium tuberculosis forms drug-tolerant persister cells that are the probable cause of its recalcitrance to antibiotic therapy. While genetically identical to the rest of the population, persisters are dormant, which protects them from killing by bactericidal antibiotics. The mechanism of persister formation in M. tuberculosis is not well understood. In this study, we selected for high persister (hip) mutants and characterized them by whole genome sequencing and transcriptome analysis. In parallel, we identified and characterized clinical isolates that naturally produce high levels of persisters. We compared the hip mutants obtained in vitro with clinical isolates to identify candidate persister genes. Genes involved in lipid biosynthesis, carbon metabolism, toxin-antitoxin systems, and transcriptional regulators were among those identified. We also found that clinical hip isolates exhibited greater ex vivo survival than the low persister isolates. Our data suggest that M. tuberculosis persister formation involves multiple pathways, and hip mutants may contribute to the recalcitrance of the infection. PMID:27176494

  2. High Persister Mutants in Mycobacterium tuberculosis

    PubMed Central

    Torrey, Heather L.; Keren, Iris; Via, Laura E.; Lee, Jong Seok; Lewis, Kim

    2016-01-01

    Mycobacterium tuberculosis forms drug-tolerant persister cells that are the probable cause of its recalcitrance to antibiotic therapy. While genetically identical to the rest of the population, persisters are dormant, which protects them from killing by bactericidal antibiotics. The mechanism of persister formation in M. tuberculosis is not well understood. In this study, we selected for high persister (hip) mutants and characterized them by whole genome sequencing and transcriptome analysis. In parallel, we identified and characterized clinical isolates that naturally produce high levels of persisters. We compared the hip mutants obtained in vitro with clinical isolates to identify candidate persister genes. Genes involved in lipid biosynthesis, carbon metabolism, toxin-antitoxin systems, and transcriptional regulators were among those identified. We also found that clinical hip isolates exhibited greater ex vivo survival than the low persister isolates. Our data suggest that M. tuberculosis persister formation involves multiple pathways, and hip mutants may contribute to the recalcitrance of the infection. PMID:27176494

  3. Peptide mimotopes of Mycobacterium tuberculosis carbohydrate immunodeterminants

    PubMed Central

    2004-01-01

    Cell-surface saccharides of Mycobacterium tuberculosis appear to be crucial factors in tuberculosis pathogenicity and could be useful antigens in tuberculosis immunodiagnosis. In the present study, we report the successful antigenic and immunogenic mimicry of mannose-containing cell-wall compounds of M. tuberculosis by dodecamer peptides identified by phage-display technology. Using a rabbit antiserum raised against M. tuberculosis cell-surface saccharides as a target for biopanning, peptides with three different consensus sequences were identified. Phage-displayed and chemically synthesized peptides bound to the anticarbohydrate antiserum. Rabbit antibodies elicited against the peptide QEPLMGTVPIRAGGGS recognize the mannosylated M. tuberculosis cell-wall antigens arabinomannan and lipoarabinomannan, and the glycosylated recombinant protein alanine/proline-rich antigen. Furthermore, antibodies were also able to react with mannan from Saccharomyces cerevisiae, but not with phosphatidylinositol dimannosides or arabinogalactan from mycobacteria. These results suggest that the immunogenic peptide mimics oligomannosidic epitopes. Interestingly, this report provides evidence that, in contrast with previously known carbohydrate mimotopes, no aromatic residues are necessary in a peptide sequence for mimicking unusual glycoconjugates synthesized by mycobacteria. The possible usefulness of the identified peptide mimotopes as surrogate reagents for immunodiagnosis and for the study of functional roles of the native non-peptide epitopes is discussed. PMID:15560754

  4. Consequences of genomic diversity in Mycobacterium tuberculosis.

    PubMed

    Coscolla, Mireia; Gagneux, Sebastien

    2014-12-01

    The causative agent of human tuberculosis, Mycobacterium tuberculosis complex (MTBC), comprises seven phylogenetically distinct lineages associated with different geographical regions. Here we review the latest findings on the nature and amount of genomic diversity within and between MTBC lineages. We then review recent evidence for the effect of this genomic diversity on mycobacterial phenotypes measured experimentally and in clinical settings. We conclude that overall, the most geographically widespread Lineage 2 (includes Beijing) and Lineage 4 (also known as Euro-American) are more virulent than other lineages that are more geographically restricted. This increased virulence is associated with delayed or reduced pro-inflammatory host immune responses, greater severity of disease, and enhanced transmission. Future work should focus on the interaction between MTBC and human genetic diversity, as well as on the environmental factors that modulate these interactions. PMID:25453224

  5. Mycobacterium tuberculosis expresses two chaperonin-60 homologs.

    PubMed Central

    Kong, T H; Coates, A R; Butcher, P D; Hickman, C J; Shinnick, T M

    1993-01-01

    A 65-kDa protein and a 10-kDa protein are two of the more strongly immunoreactive components of Mycobacterium tuberculosis, the causative agent of tuberculosis. The 65-kDa antigen has homology with members of the GroEL or chaperonin-60 (Cpn60) family of heat shock proteins. The 10-kDa antigen has homology with the GroES or chaperonin-10 family of heat shock proteins. These two proteins are encoded by separate genes in M. tuberculosis. The studies reported here reveal that M. tuberculosis contains a second Cpn60 homolog located 98 bp downstream of the 10-kDa antigen gene. The second Cpn60 homolog (Cpn60-1) displays 61% amino acid sequence identity with the 65-kDa antigen (Cpn60-2) and 53% and 41% identity with the Escherichia coli GroEL protein and the human P60 protein, respectively. Primer-extension analysis revealed that transcription starts 29 bp upstream of the translation start of the Cpn60-1 homolog and protein purification studies indicate that the cpn60-1 gene is expressed as an approximately 60-kDa polypeptide. Images Fig. 3 Fig. 5 PMID:7681982

  6. Mycobacterium tuberculosis is resistant to streptolydigin.

    PubMed

    Speer, Alexander; Rowland, Jennifer L; Niederweis, Michael

    2013-07-01

    Drug resistant strains of Mycobacterium tuberculosis (Mtb) undermine tuberculosis (TB) control. Streptolydigin is a broadly effective antibiotic which inhibits RNA polymerase, similarly to rifampicin, a key drug in current TB chemotherapeutic regimens. Due to a vastly improved chemical synthesis streptolydigin and derivatives are being promoted as putative TB drugs. The microplate Alamar Blue assay revealed that Streptococcus salivarius and Mycobacterium smegmatis were susceptible to streptolydigin with minimum inhibitory concentrations (MICs) of 1.6 mg/L and 6.25 mg/L, respectively. By contrast, the MICs of streptolydigin and two derivatives, streptolydiginone and dihydrostreptolydigin, against Mtb were ≥ 100 mg/L demonstrating that Mtb is resistant to streptolydigin in contrast to previous reports. Further, a porin mutant of M. smegmatis is resistant to streptolydigin indicating that porins mediate uptake of streptolydigin across the outer membrane. Since the RNA polymerase is a validated drug target in Mtb and porins are required for susceptibility of M. smegmatis, the absence of MspA-like porins probably contributes to the resistance of Mtb to streptolydigin. This study shows that streptolydigin is not a suitable drug in TB treatment regimens. PMID:23591156

  7. Tenosynovitis caused by a novel nontuberculous Mycobacterium species initially misidentified as a member of the Mycobacterium tuberculosis complex.

    PubMed

    Simner, Patricia J; Hyle, Emily P; Buckwalter, Seanne P; Branda, John A; Brown-Elliott, Barbara A; Franklin, Jameelah; Toney, Nadege C; de Man, Tom J B; Wallace, Richard J; Vasireddy, Ravikiran; Gandhi, Rajesh T; Wengenack, Nancy L

    2014-12-01

    We present a case of tenosynovitis caused by a novel, slowly growing, nonchromogenic, nontuberculous mycobacterium (NTM). Originally misidentified as Mycobacterium tuberculosis complex, the NTM cross-reacts with the M. tuberculosis complex nucleic acid hybridization probe, a M. tuberculosis gamma interferon release assay, and is closely related to M. tuberculosis by 16S rRNA gene sequencing. PMID:25253791

  8. The 65-kilodalton antigen of Mycobacterium tuberculosis.

    PubMed Central

    Shinnick, T M

    1987-01-01

    The immune response of the host to the antigens of Mycobacterium tuberculosis plays the key role in determining immunity from infection with as well as the pathogenicity of this organism. A 65-kilodalton (kDa) protein has been identified as one of the medically important antigens of M. tuberculosis. The gene encoding this antigen was isolated from a lambda gt11-M. tuberculosis recombinant DNA library using monoclonal antibodies directed against the 65-kDa antigen as the specific probes. The nucleotide sequence of this gene was determined, and a 540-amino-acid sequence was deduced. This sequence was shown to correspond to that of the 65-kDa antigen by constructing a plasmid in which this open reading frame was fused to the lacZ gene. The resulting fusion protein reacted specifically with the anti-65-kDa protein antibodies. A second long open reading frame was found downstream of the 65-kDa antigen gene which could encode a protein of 517 amino acids. This putative protein contained 29 tandemly arranged partial or complete matches to a pentapeptide sequence. Images PMID:3029018

  9. Genome Sequencing and Annotation of Mycobacterium tuberculosis PR08 strain.

    PubMed

    Jaafar, Mohammad Maaruf; Halim, Mohd Zakihalani A; Ismail, Mohamad Izwan; Shien, Lee Lian; Kek, Teh Lay; Fong, Ngeow Yun; Nor, Norazmi Mohd; Zainuddin, Zainul Fadziruddin; Hock, Tang Thean; Najimudin, Mohd Nazalan Mohd; Salleh, Mohd Zaki

    2016-03-01

    Mycobacterium tuberculosis is an acid fast bacterial species in the family Mycobacteriaceae and is the causative agent of most cases of tuberculosis. Here, we report the genomic features of Mycobacterium tuberculosis isolated from the cerebrospinal fluid (CSF) of a patient diagnosed with both pulmonary and extrapulmonary tuberculosis (TB). The isolated strain was identified as Mycobacterium tuberculosis PR08 (MTB PR08). Genomic DNA of the MTB PR08 strain was extracted and subjected to whole genome sequencing using MiSeq (Illumina, CA,USA). The draft genome size of MTB PR08 strain is 4,292,364 bp with a G + C content of 65.2%. This strain was annotated to have 4723 genes and 48 RNAs. This whole genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession number CP010895. PMID:26981383

  10. Mycobacterium tuberculosis supports protein tyrosine phosphorylation

    PubMed Central

    Kusebauch, Ulrike; Ortega, Corrie; Ollodart, Anja; Rogers, Richard S.; Sherman, David R.; Moritz, Robert L.; Grundner, Christoph

    2014-01-01

    Reversible protein phosphorylation determines growth and adaptive decisions in Mycobacterium tuberculosis (Mtb). At least 11 two-component systems and 11 Ser/Thr protein kinases (STPKs) mediate phosphorylation on Asp, His, Ser, and Thr. In contrast, protein phosphorylation on Tyr has not been described previously in Mtb. Here, using a combination of phospho-enrichment and highly sensitive mass spectrometry, we show extensive protein Tyr phosphorylation of diverse Mtb proteins, including STPKs. Several STPKs function as dual-specificity kinases that phosphorylate Tyr in cis and in trans, suggesting that dual-specificity kinases have a major role in bacterial phospho-signaling. Mutation of a phosphotyrosine site of the essential STPK PknB reduces its activity in vitro and in live Mtb, indicating that Tyr phosphorylation has a functional role in bacterial growth. These data identify a previously unrecognized phosphorylation system in a human pathogen that claims ∼1.4 million lives every year. PMID:24927537

  11. Characterization of Mycobacterium orygis as M. tuberculosis complex subspecies.

    PubMed

    van Ingen, Jakko; Rahim, Zeaur; Mulder, Arnout; Boeree, Martin J; Simeone, Roxane; Brosch, Roland; van Soolingen, Dick

    2012-04-01

    The oryx bacilli are Mycobacterium tuberculosis complex organisms for which phylogenetic position and host range are unsettled. We characterized 22 isolates by molecular methods and propose elevation to subspecies status as M. orygis. M. orygis is a causative agent of tuberculosis in animals and humans from Africa and South Asia. PMID:22469053

  12. The cell envelope glycoconjugates of Mycobacterium tuberculosis

    PubMed Central

    Angala, Shiva Kumar; Belardinelli, Juan Manuel; Huc-Claustre, Emilie; Wheat, William H.; Jackson, Mary

    2015-01-01

    Tuberculosis (TB) remains the second most common cause of death due to a single infectious agent. The cell envelope of Mycobacterium tuberculosis (Mtb), the causative agent of the disease in humans, is a source of unique glycoconjugates and the most distinctive feature of the biology of this organism. It is the basis of much of Mtb pathogenesis and one of the major causes of its intrinsic resistance to chemotherapeutic agents. At the same time, the unique structures of Mtb cell envelope glycoconjugates, their antigenicity and essentiality for mycobacterial growth provide opportunities for drug, vaccine, diagnostic and biomarker development, as clearly illustrated by recent advances in all of these translational aspects. This review focuses on our current understanding of the structure and biogenesis of Mtb glycoconjugates with particular emphasis on one of most intriguing and least understood aspect of the physiology of mycobacteria: the translocation of these complex macromolecules across the different layers of the cell envelope. It further reviews the rather impressive progress made in the last ten years in the discovery and development of novel inhibitors targeting their biogenesis. PMID:24915502

  13. Human immune response to Mycobacterium tuberculosis antigens.

    PubMed Central

    Havlir, D V; Wallis, R S; Boom, W H; Daniel, T M; Chervenak, K; Ellner, J J

    1991-01-01

    Little is known about the immunodominant or protective antigens of Mycobacterium tuberculosis in humans. Cell-mediated immunity is necessary for protection, and healthy tuberculin-positive individuals are relatively resistant to exogenous reinfection. We compared the targets of the cell-mediated immune response in healthy tuberculin-positive individuals to those of tuberculosis patients and tuberculin-negative persons. By using T-cell Western blotting (immunoblotting) of nitrocellulose-bound M. tuberculosis culture filtrate, peaks of T-cell blastogenic activity were identified in the healthy tuberculin reactors at 30, 37, 44, 57, 64, 71 and 88 kDa. Three of these fractions (30, 64, and 71 kDa) coincided with previously characterized proteins: antigen 6/alpha antigen, HSP60, and HSP70, respectively. The blastogenic responses to purified M. tuberculosis antigen 6/alpha antigen and BCG HSP60 were assessed. When cultured with purified antigen 6/alpha antigen, lymphocytes of healthy tuberculin reactors demonstrated greater [3H]thymidine incorporation than either healthy tuberculin-negative controls or tuberculous patients (8,113 +/- 1,939 delta cpm versus 645 +/- 425 delta cpm and 1,019 +/- 710 delta cpm, respectively; P less than 0.01). Healthy reactors also responded to HSP60, although to a lesser degree than antigen 6/alpha antigen (4,276 +/- 1,095 delta cpm; P less than 0.05). Partially purified HSP70 bound to nitrocellulose paper elicited a significant lymphocyte blastogenic response in two of six of the tuberculous patients but in none of the eight healthy tuberculin reactors. Lymphocytes of none of five tuberculin-negative controls responded to recombinant antigens at 14 or 19 kDa or to HSP70. Antibody reactivity generally was inversely correlated with blastogenic response: tuberculous sera had high titer antibody to M. tuberculosis culture filtrate in a range from 35 to 180 kDa. This is the first systematic evaluation of the human response to a panel of native

  14. Fate of Mycobacterium tuberculosis inside rat peritoneal macrophages in vitro.

    PubMed

    Vishwanath, V; Meera, R; Puvanakrishnan, R; Narayanan, P R

    1997-10-01

    Rat peritoneal macrophages in vitro were infected with Mycobacterium tuberculosis and the fate of M. tuberculosis inside macrophages was monitored. Alteration in the levels of nitric oxide (NO) measured in terms of nitrite formed, hydrogen peroxide (H2O2) and lysosomal enzymes such as acid phosphatase, cathepsin-D and beta-glucuronidase in macrophages following M. tuberculosis infection was also studied. Elevation in the levels of nitrite were observed from 72 h of M. tuberculosis infection. Irrespective of the time point, M. tuberculosis infected macrophages produced elevated levels of H2O2. Maximum increase in the level of acid phosphatase was observed from 72 h of M. tuberculosis infection, whereas maximum elevation in the level of beta-glucuronidase was observed 48 h after M. tuberculosis infection. However these microbicidal agents did not alter the intracellular viability of M. tuberculosis. PMID:9350049

  15. Insights from the complete genome sequence of Mycobacterium marinum on the evolution of Mycobacterium tuberculosis

    PubMed Central

    Stinear, Timothy P.; Seemann, Torsten; Harrison, Paul F.; Jenkin, Grant A.; Davies, John K.; Johnson, Paul D.R.; Abdellah, Zahra; Arrowsmith, Claire; Chillingworth, Tracey; Churcher, Carol; Clarke, Kay; Cronin, Ann; Davis, Paul; Goodhead, Ian; Holroyd, Nancy; Jagels, Kay; Lord, Angela; Moule, Sharon; Mungall, Karen; Norbertczak, Halina; Quail, Michael A.; Rabbinowitsch, Ester; Walker, Danielle; White, Brian; Whitehead, Sally; Small, Pamela L.C.; Brosch, Roland; Ramakrishnan, Lalita; Fischbach, Michael A.; Parkhill, Julian; Cole, Stewart T.

    2008-01-01

    Mycobacterium marinum, a ubiquitous pathogen of fish and amphibia, is a near relative of Mycobacterium tuberculosis, the etiologic agent of tuberculosis in humans. The genome of the M strain of M. marinum comprises a 6,636,827-bp circular chromosome with 5424 CDS, 10 prophages, and a 23-kb mercury-resistance plasmid. Prominent features are the very large number of genes (57) encoding polyketide synthases (PKSs) and nonribosomal peptide synthases (NRPSs) and the most extensive repertoire yet reported of the mycobacteria-restricted PE and PPE proteins, and related-ESX secretion systems. Some of the NRPS genes comprise a novel family and seem to have been acquired horizontally. M. marinum is used widely as a model organism to study M. tuberculosis pathogenesis, and genome comparisons confirmed the close genetic relationship between these two species, as they share 3000 orthologs with an average amino acid identity of 85%. Comparisons with the more distantly related Mycobacterium avium subspecies paratuberculosis and Mycobacterium smegmatis reveal how an ancestral generalist mycobacterium evolved into M. tuberculosis and M. marinum. M. tuberculosis has undergone genome downsizing and extensive lateral gene transfer to become a specialized pathogen of humans and other primates without retaining an environmental niche. M. marinum has maintained a large genome so as to retain the capacity for environmental survival while becoming a broad host range pathogen that produces disease strikingly similar to M. tuberculosis. The work described herein provides a foundation for using M. marinum to better understand the determinants of pathogenesis of tuberculosis. PMID:18403782

  16. Mycobacterium marinum: a potential immunotherapy for Mycobacterium tuberculosis infection

    PubMed Central

    Tian, Wei-wei; Wang, Qian-qiu; Liu, Wei-da; Shen, Jian-ping; Wang, Hong-sheng

    2013-01-01

    Purpose The aim of the present study was to investigate the immune response induced by Mycobacterium marinum infection in vitro and the potential of M. marinum as an immunotherapy for M. tuberculosis infection. Methods The potential human immune response to certain bacillus infections was investigated in an immune cell-bacillus coculture system in vitro. As a potential novel immunotherapy, M. marinum was studied and compared with two other bacilli, Bacillus Calmette-Guérin (BCG) and live attenuated M. tuberculosis. We examined the changes in both the bacilli and immune cells, especially the time course of the viability of mycobacteria in the coculture system and host immune responses including multinuclear giant cell formation by Wright-Giemsa modified staining, macrophage polarization by cell surface antigen expression, and cytokines/chemokine production by both mRNA expression and protein secretion. Results The M. marinum stimulated coculture group showed more expression of CD209, CD68, CD80, and CD86 than the BCG and M. tuberculosis (an attenuated strain, H37Ra) groups, although the differences were not statistically significant. Moreover, the M. marinum group expressed more interleukin (IL)-1B and IL-12p40 on day 3 (IL-1B: P = 0.003 and 0.004, respectively; IL-12p40: P = 0.001 and 0.011, respectively), a higher level of CXCL10 on day 1 (P = 0.006 and 0.026, respectively), and higher levels of chemokine (C-X-C motif) ligand (CXCL) 8 and chemokine (C motif) ligand (XCL) 1 on day 3 (CXCL8: P = 0.012 and 0.014, respectively; XCL1: P = 0.000 and 0.000, respectively). The M. marinum stimulated coculture group also secreted more tumor necrosis factor (TNF)-α, IL-1β, and IL-10 on day 1 (TNF-α: P = 0.000 and 0.000, respectively; IL-1β: P = 0.000 and 0.000, respectively; IL-10: P = 0.002 and 0.019, respectively) and day 3 (TNF-α: P = 0.000 and 0.000, respectively; IL-1β: P = 0.000 and 0.001, respectively; IL-10: P = 0.000 and 0.000, respectively). In addition, the

  17. Evaluation of the results of Mycobacterium tuberculosis direct test (MTD) and Mycobacterial culture in urine samples

    PubMed Central

    Sener, Asli Gamze; Kurultay, Nukhet; Afsar, Ilhan

    2008-01-01

    Tuberculosis remains a public health problem in Turkey. Rapid detection of Mycobacterium tuberculosis plays a key role in control of infection. In this article, the Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test (MTD) was evaluated for detection of M. tuberculosis in urine samples. The performance of the MTD was very good and appropriate for routine laboratory diagnosis. PMID:24031287

  18. Characterization of Lipoyl Synthase from Mycobacterium tuberculosis.

    PubMed

    Lanz, Nicholas D; Lee, Kyung-Hoon; Horstmann, Abigail K; Pandelia, Maria-Eirini; Cicchillo, Robert M; Krebs, Carsten; Booker, Squire J

    2016-03-01

    The prevalence of multiple and extensively drug-resistant strains of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is on the rise, necessitating the identification of new targets to combat an organism that has infected one-third of the world's population, according to the World Health Organization. The biosynthesis of the lipoyl cofactor is one possible target, given its critical importance in cellular metabolism and the apparent lack of functional salvage pathways in Mtb that are found in humans and many other organisms. The lipoyl cofactor is synthesized de novo in two committed steps, involving the LipB-catalyzed transfer of an octanoyl chain derived from fatty acid biosynthesis to a lipoyl carrier protein and the LipA-catalyzed insertion of sulfur atoms at C6 and C8 of the octanoyl chain. A number of in vitro studies of lipoyl synthases from Escherichia coli, Sulfolobus solfataricus, and Thermosynechococcus elongatus have been conducted, but the enzyme from Mtb has not been characterized. Herein, we show that LipA from Mtb contains two [4Fe-4S] clusters and converts an octanoyl peptide substrate to the corresponding lipoyl peptide product via the same C6-monothiolated intermediate as that observed in the E. coli LipA reaction. In addition, we show that LipA from Mtb forms a complex with the H protein of the glycine cleavage system and that the strength of association is dependent on the presence of S-adenosyl-l-methionine. We also show that LipA from Mtb can complement a lipA mutant of E. coli, demonstrating the commonalities of the two enzymes. Lastly, we show that the substrate for LipA, which normally acts on a post-translationally modified protein, can be reduced to carboxybenzyl-octanoyllysine. PMID:26841001

  19. Pulmonary disease due to Mycobacterium tuberculosis in a horse: zoonotic concerns and limitations of antemortem testing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A case of pulmonary tuberculosis caused by Mycobacterium tuberculosis was diagnosed in a horse. Clinical evaluation performed prior to euthanasia did not suggest tuberculosis, but postmortem examination provided pathological and bacteriological evidence of disease. In the lungs, multiple tuberculoid...

  20. MTBreg: The Database of Conditionally Regulated Proteins in Mycobacterium Tuberculosis

    DOE Data Explorer

    Kaufman, Markus; Pal, Debnath; Eisenberg, David

    Proteins up- and down- regulated in Mycobacterium tuberculosis grown under conditions mimicking infection are included in this database. It also includes information on proteins that are regulated by selected transcription factors or other regulatory proteins. The literature data provided here is complimentary to the databases provided by Michael Strong that include recent TB computational functional linkages and the Prolinks Database by Peter Bowers. The experimental condition, the experimental dataset and a literature reference will be displayed, including links to the computationally linked proteins in the Prolinks Database and the entry in the Mycobacterium tuberculosis Structural Genomics Database.[Copied from information at http://www.doe-mbi.ucla.edu/Services/MTBreg/

  1. Two Cases of Pulmonary Tuberculosis Caused by Mycobacterium tuberculosis subsp. canetti

    PubMed Central

    Morillon, Marc; Koeck, Jean-Louis; Varnerot, Anne; Briant, Jean-François; Nguyen, Gilbert; Verrot, Denis; Bonnet, Daniel; Vincent, Véronique

    2002-01-01

    We identified an unusual strain of mycobacteria from two patients with pulmonary tuberculosis by its smooth, glossy morphotype and, primarily, its genotypic characteristics. Spoligotyping and restriction fragment length polymorphism typing were carried out with the insertion sequence IS6110 patterns. All known cases of tuberculosis caused by Mycobacterium canetti have been contracted in the Horn of Africa. PMID:12453369

  2. Human Xenobiotic Nuclear Receptor PXR Augments Mycobacterium tuberculosis Survival.

    PubMed

    Bhagyaraj, Ella; Nanduri, Ravikanth; Saini, Ankita; Dkhar, Hedwin Kitdorlang; Ahuja, Nancy; Chandra, Vemika; Mahajan, Sahil; Kalra, Rashi; Tiwari, Drishti; Sharma, Charu; Janmeja, Ashok Kumar; Gupta, Pawan

    2016-07-01

    Mycobacterium tuberculosis can evade host defense processes, thereby ensuring its survival and pathogenesis. In this study, we investigated the role of nuclear receptor, pregnane X receptor (PXR), in M. tuberculosis infection in human monocyte-derived macrophages. In this study, we demonstrate that PXR augments M. tuberculosis survival inside the host macrophages by promoting the foamy macrophage formation and abrogating phagolysosomal fusion, inflammation, and apoptosis. Additionally, M. tuberculosis cell wall lipids, particularly mycolic acids, crosstalk with human PXR (hPXR) by interacting with its promiscuous ligand binding domain. To confirm our in vitro findings and to avoid the reported species barrier in PXR function, we adopted an in vivo mouse model expressing hPXR, wherein expression of hPXR in mice promotes M. tuberculosis survival. Therefore, pharmacological intervention and designing antagonists to hPXR may prove to be a promising adjunct therapy for tuberculosis. PMID:27233963

  3. Dramatic reduction of culture time of Mycobacterium tuberculosis

    NASA Astrophysics Data System (ADS)

    Ghodbane, Ramzi; Raoult, Didier; Drancourt, Michel

    2014-02-01

    Mycobacterium tuberculosis culture, a critical technique for routine diagnosis of tuberculosis, takes more than two weeks. Here, step-by-step improvements in the protocol including a new medium, microaerophlic atmosphere or ascorbic-acid supplement and autofluorescence detection dramatically shortened this delay. In the best case, primary culture and rifampicin susceptibility testing were achieved in 72 hours when specimens were inoculated directly on the medium supplemented by antibiotic at the beginning of the culture.

  4. Genomic signal analysis of Mycobacterium tuberculosis

    NASA Astrophysics Data System (ADS)

    Cristea, Paul Dan; Banica, Dorina; Tuduce, Rodica

    2007-02-01

    As previously shown the conversion of nucleotide sequences into digital signals offers the possibility to apply signal processing methods for the analysis of genomic data. Genomic Signal Analysis (GSA) has been used to analyze large scale features of DNA sequences, at the scale of whole chromosomes, including both coding and non-coding regions. The striking regularities of genomic signals reveal restrictions in the way nucleotides and pairs of nucleotides are distributed along nucleotide sequences. Structurally, a chromosome appears to be less of a "plain text", corresponding to certain semantic and grammar rules, but more of a "poem", satisfying additional symmetry restrictions that evoke the "rhythm" and "rhyme". Recurrent patterns in nucleotide sequences are reflected in simple mathematical regularities observed in genomic signals. GSA has also been used to track pathogen variability, especially concerning their resistance to drugs. Previous work has been dedicated to the study of HIV-1, Clade F and Avian Flu. The present paper applies GSA methodology to study Mycobacterium tuberculosis (MT) rpoB gene variability, relevant to its resistance to antibiotics. Isolates from 50 Romanian patients have been studied both by rapid LightCycler PCR and by sequencing of a segment of 190-250 nucleotides covering the region of interest. The variability is caused by SNPs occurring at specific sites along the gene strand, as well as by inclusions. Because of the mentioned symmetry restrictions, the GS variations tend to compensate. An important result is that MT can act as a vector for HIV virus, which is able to retrotranscribe its specific genes both into human and MT genomes.

  5. Detection of Mycobacterium tuberculosis in clinical specimens by polymerase chain reaction and Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test.

    PubMed Central

    Abe, C; Hirano, K; Wada, M; Kazumi, Y; Takahashi, M; Fukasawa, Y; Yoshimura, T; Miyagi, C; Goto, S

    1993-01-01

    The polymerase chain reaction (PCR) using oligonucleotides based on the repetitive sequence (IS986) of Mycobacterium tuberculosis as a primer and the Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test (MTD), which combines an M. tuberculosis rRNA amplification method with the hybridization protection assay format, were evaluated for detection of M. tuberculosis in clinical samples. The detection limits of these two assay systems based on nucleic acid amplification for cultured M. tuberculosis were less than 10 cells per reaction. A total of 135 sputum specimens were examined by the two assay systems. The PCR and the MTD systems for detection of M. tuberculosis gave overall positivity rates of 84.2% (32 of 38) and 91.9% (34 of 37), respectively, as compared with 71.9% (23 of 32) by smear and 96.9% (31 of 32) by culture in the liquid medium MB-Check. Procedures for sample preparation used in the two methods were different. Although the sensitivities of the PCR and MTD appeared to be similar to that of culture with the MB-Check system, the two methods based on nucleic acid amplification should be very useful for rapid detection of M. tuberculosis infections without the long time required for culture of M. tuberculosis. Images PMID:8308121

  6. Novel Cephalosporins Selectively Active on Nonreplicating Mycobacterium tuberculosis

    PubMed Central

    2016-01-01

    We report two series of novel cephalosporins that are bactericidal to Mycobacterium tuberculosis alone of the pathogens tested, which only kill M. tuberculosis when its replication is halted by conditions resembling those believed to pertain in the host, and whose bactericidal activity is not dependent upon or enhanced by clavulanate, a β-lactamase inhibitor. The two classes of cephalosporins bear an ester or alternatively an oxadiazole isostere at C-2 of the cephalosporin ring system, a position that is almost exclusively a carboxylic acid in clinically used agents in the class. Representatives of the series kill M. tuberculosis within macrophages without toxicity to the macrophages or other mammalian cells. PMID:27144688

  7. Novel Cephalosporins Selectively Active on Nonreplicating Mycobacterium tuberculosis.

    PubMed

    Gold, Ben; Smith, Robert; Nguyen, Quyen; Roberts, Julia; Ling, Yan; Lopez Quezada, Landys; Somersan, Selin; Warrier, Thulasi; Little, David; Pingle, Maneesh; Zhang, David; Ballinger, Elaine; Zimmerman, Matthew; Dartois, Véronique; Hanson, Paul; Mitscher, Lester A; Porubsky, Patrick; Rogers, Steven; Schoenen, Frank J; Nathan, Carl; Aubé, Jeffrey

    2016-07-14

    We report two series of novel cephalosporins that are bactericidal to Mycobacterium tuberculosis alone of the pathogens tested, which only kill M. tuberculosis when its replication is halted by conditions resembling those believed to pertain in the host, and whose bactericidal activity is not dependent upon or enhanced by clavulanate, a β-lactamase inhibitor. The two classes of cephalosporins bear an ester or alternatively an oxadiazole isostere at C-2 of the cephalosporin ring system, a position that is almost exclusively a carboxylic acid in clinically used agents in the class. Representatives of the series kill M. tuberculosis within macrophages without toxicity to the macrophages or other mammalian cells. PMID:27144688

  8. Co-evolution of Mycobacterium tuberculosis and Homo sapiens

    PubMed Central

    Brites, Daniela; Gagneux, Sebastien

    2015-01-01

    The causative agent of human tuberculosis (TB), Mycobacterium tuberculosis, is an obligate pathogen that evolved to exclusively persist in human populations. For M. tuberculosis to transmit from person to person, it has to cause pulmonary disease. Therefore, M. tuberculosis virulence has likely been a significant determinant of the association between M. tuberculosis and humans. Indeed, the evolutionary success of some M. tuberculosis genotypes seems at least partially attributable to their increased virulence. The latter possibly evolved as a consequence of human demographic expansions. If co-evolution occurred, humans would have counteracted to minimize the deleterious effects of M. tuberculosis virulence. The fact that human resistance to infection has a strong genetic basis is a likely consequence of such a counter-response. The genetic architecture underlying human resistance to M. tuberculosis remains largely elusive. However, interactions between human genetic polymorphisms and M. tuberculosis genotypes have been reported. Such interactions are consistent with local adaptation and allow for a better understanding of protective immunity in TB. Future ‘genome-to-genome’ studies, in which locally associated human and M. tuberculosis genotypes are interrogated in conjunction, will help identify new protective antigens for the development of better TB vaccines. PMID:25703549

  9. Can Molecular Methods Detect 1% Isoniazid Resistance in Mycobacterium tuberculosis?

    PubMed Central

    Folkvardsen, Dorte Bek; Thomsen, Vibeke Ø.; Rasmussen, Erik Michael; Bang, Didi; Werngren, Jim; Hoffner, Sven; Hillemann, Doris; Rigouts, Leen

    2013-01-01

    Patients may harbor both drug-susceptible and -resistant bacteria, representing heteroresistance. We studied mixtures of isoniazid-resistant and -susceptible Mycobacterium tuberculosis strains. Conventional drug susceptibility testing was the most sensitive method of detection, whereas the line probe assay and sequencing were not able to detect the clinically relevant 1% proportion of resistant bacteria. PMID:23447641

  10. A case of Manila type Mycobacterium tuberculosis infection in Japan

    PubMed Central

    Usami, Osamu; Nakajima, Chie; Endo, Shiro; Inomata, Shinya; Kanamori, Hajime; Hirakata, Yoichi; Uchiyama, Bine; Kaku, Mitsuo; Suzuki, Yasuhiko; Hattori, Toshio

    2015-01-01

    Key Clinical Message A 76-year-old Japanese woman contracted a Mycobacterium tuberculosis (TB, Manila type) infection in Japan, despite never having traveled. However, her son was treated for TB in the Philippines 3 years before he stayed at her house. Spoligotyping allows us to identify the TB genotype and identify the route of infection. PMID:26273455

  11. 21 CFR 866.3370 - Mycobacterium tuberculosis immunofluorescent reagents.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Mycobacterium tuberculosis immunofluorescent reagents. 866.3370 Section 866.3370 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological...

  12. 21 CFR 866.3370 - Mycobacterium tuberculosis immunofluorescent reagents.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Mycobacterium tuberculosis immunofluorescent reagents. 866.3370 Section 866.3370 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological...

  13. 21 CFR 866.3370 - Mycobacterium tuberculosis immunofluorescent reagents.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Mycobacterium tuberculosis immunofluorescent reagents. 866.3370 Section 866.3370 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological...

  14. 21 CFR 866.3370 - Mycobacterium tuberculosis immunofluorescent reagents.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Mycobacterium tuberculosis immunofluorescent reagents. 866.3370 Section 866.3370 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological...

  15. 21 CFR 866.3370 - Mycobacterium tuberculosis immunofluorescent reagents.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Mycobacterium tuberculosis immunofluorescent reagents. 866.3370 Section 866.3370 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological...

  16. Insights into redox sensing metalloproteins in Mycobacterium tuberculosis.

    PubMed

    Chim, Nicholas; Johnson, Parker M; Goulding, Celia W

    2014-04-01

    Mycobacterium tuberculosis, the pathogen that causes tuberculosis, has evolved sophisticated mechanisms for evading assault by the human host. This review focuses on M. tuberculosis regulatory metalloproteins that are sensitive to exogenous stresses attributed to changes in the levels of gaseous molecules (i.e., molecular oxygen, carbon monoxide and nitric oxide) to elicit an intracellular response. In particular, we highlight recent developments on the subfamily of Whi proteins, redox sensing WhiB-like proteins that contain iron-sulfur clusters, sigma factors and their cognate anti-sigma factors of which some are zinc-regulated, and the dormancy survival regulon DosS/DosT-DosR heme sensory system. Mounting experimental evidence suggests that these systems contribute to a highly complex and interrelated regulatory network that controls M. tuberculosis biology. This review concludes with a discussion of strategies that M. tuberculosis has developed to maintain redox homeostasis, including mechanisms to regulate endogenous nitric oxide and carbon monoxide levels. PMID:24314844

  17. Biosensing Technologies for Mycobacterium tuberculosis Detection: Status and New Developments

    PubMed Central

    Zhou, Lixia; He, Xiaoxiao; He, Dinggeng; Wang, Kemin; Qin, Dilan

    2011-01-01

    Biosensing technologies promise to improve Mycobacterium tuberculosis (M. tuberculosis) detection and management in clinical diagnosis, food analysis, bioprocess, and environmental monitoring. A variety of portable, rapid, and sensitive biosensors with immediate “on-the-spot” interpretation have been developed for M. tuberculosis detection based on different biological elements recognition systems and basic signal transducer principles. Here, we present a synopsis of current developments of biosensing technologies for M. tuberculosis detection, which are classified on the basis of basic signal transducer principles, including piezoelectric quartz crystal biosensors, electrochemical biosensors, and magnetoelastic biosensors. Special attention is paid to the methods for improving the framework and analytical parameters of the biosensors, including sensitivity and analysis time as well as automation of analysis procedures. Challenges and perspectives of biosensing technologies development for M. tuberculosis detection are also discussed in the final part of this paper. PMID:21437177

  18. Rapid diagnosis of Mycobacterium tuberculosis bacteremia by PCR.

    PubMed Central

    Folgueira, L; Delgado, R; Palenque, E; Aguado, J M; Noriega, A R

    1996-01-01

    A method based on DNA amplification and hybridization has been used for the rapid detection of Mycobacterium tuberculosis in blood samples from 38 hospitalized patients (15 human immunodeficiency virus [HIV] positive and 23 HIV negative) in whom localized or disseminated forms of tuberculosis were suspected. In 32 of these patients, the diagnosis of tuberculosis was eventually confirmed by conventional bacteriological or histological procedures. M. tuberculosis DNA was detected with the PCR technique in the peripheral blood mononuclear cells from 9 of 11 (82%) HIV-infected patients and in 7 of 21 (33%) HIV-negative patients (P < 0.01), while M. tuberculosis blood cultures were positive in 1 of 8 (12.5%) and 1 of 18 (5.5%) patients, respectively. PCR was positive in all cases with disseminated disease in both HIV-negative and HIV-positive patients and also in the HIV-positive patients with extrapulmonary tuberculosis. Seven samples from patients with documented illness other than tuberculosis and 12 specimens from healthy volunteers, including seven volunteers with a recent positive purified protein derivative test, were used as controls and had a negative PCR. These results suggest that detection of M. tuberculosis DNA in peripheral blood mononuclear cells may be a useful tool for rapid diagnosis of disseminated and extrapulmonary forms of tuberculosis, especially in an HIV-positive population. PMID:8904404

  19. Mycobacterium tuberculosis resistance to antituberculosis drugs in Mozambique*, **

    PubMed Central

    Pires, Germano Manuel; Folgosa, Elena; Nquobile, Ndlovu; Gitta, Sheba; Cadir, Nureisha

    2014-01-01

    OBJECTIVE: To determine the drug resistance profile of Mycobacterium tuberculosis in Mozambique. METHODS: We analyzed secondary data from the National Tuberculosis Referral Laboratory, in the city of Maputo, Mozambique, and from the Beira Regional Tuberculosis Referral Laboratory, in the city of Beira, Mozambique. The data were based on culture-positive samples submitted to first-line drug susceptibility testing (DST) between January and December of 2011. We attempted to determine whether the frequency of DST positivity was associated with patient type or provenance. RESULTS: During the study period, 641 strains were isolated in culture and submitted to DST. We found that 374 (58.3%) were resistant to at least one antituberculosis drug and 280 (43.7%) were resistant to multiple antituberculosis drugs. Of the 280 multidrug-resistant tuberculosis cases, 184 (65.7%) were in previously treated patients, most of whom were from southern Mozambique. Two (0.71%) of the cases of multidrug-resistant tuberculosis were confirmed to be cases of extensively drug-resistant tuberculosis. Multidrug-resistant tuberculosis was most common in males, particularly those in the 21-40 year age bracket. CONCLUSIONS: M. tuberculosis resistance to antituberculosis drugs is high in Mozambique, especially in previously treated patients. The frequency of M. tuberculosis strains that were resistant to isoniazid, rifampin, and streptomycin in combination was found to be high, particularly in samples from previously treated patients. PMID:24831398

  20. Proteome Analysis of the Plasma Membrane of Mycobacterium Tuberculosis

    PubMed Central

    Arora, Shalini; Kosalai, K.; Namane, Abdelkader; Pym, Alex S.; Cole, Stewart T.

    2002-01-01

    The plasma membrane of Mycobacterium tuberculosis is likely to contain proteins that could serve as novel drug targets, diagnostic probes or even components of a vaccine against tuberculosis. With this in mind, we have undertaken proteome analysis of the membrane of M. tuberculosis H37Rv. Isolated membrane vesicles were extracted with either a detergent (Triton X114) or an alkaline buffer (carbonate) following two of the protocols recommended for membrane protein enrichment. Proteins were resolved by 2D-GE using immobilized pH gradient (IPG) strips, and identified by peptide mass mapping utilizing the M. tuberculosis genome database. The two extraction procedures yielded patterns with minimal overlap. Only two proteins, both HSPs, showed a common presence. MALDI–MS analysis of 61 spots led to the identification of 32 proteins, 17 of which were new to the M. tuberculosis proteome database. We classified 19 of the identified proteins as ‘membrane-associated’; 14 of these were further classified as ‘membrane-bound’, three of which were lipoproteins. The remaining proteins included four heat-shock proteins and several enzymes involved in energy or lipid metabolism. Extraction with Triton X114 was found to be more effective than carbonate for detecting ‘putative’ M. tuberculosis membrane proteins. The protocol was also found to be suitable for comparing BCG and M. tuberculosis membranes, identifying ESAT-6 as being expressed selectively in M. tuberculosis. While this study demonstrates for the first time some of the membrane proteins of M. tuberculosis, it also underscores the problems associated with proteomic analysis of a complex membrane such as that of a mycobacterium. PMID:18629250

  1. Determination of Urinary Neopterin/Creatinine Ratio to Distinguish Active Tuberculosis from Latent Mycobacterium tuberculosis Infection

    PubMed Central

    Eisenhut, Michael; Hargreaves, Dougal S.; Scott, Anne; Housley, David; Walters, Andrew; Mulla, Rohinton

    2016-01-01

    Background. Biomarkers to distinguish latent from active Mycobacterium (M.) tuberculosis infection in clinical practice are lacking. The urinary neopterin/creatinine ratio can quantify the systemic interferon-gamma effect in patients with M. tuberculosis infection. Methods. In a prospective observational study, urinary neopterin levels were measured by enzyme linked immunosorbent assay in patients with active tuberculosis, in people with latent M. tuberculosis infection, and in healthy controls and the urinary neopterin/creatinine ratio was calculated. Results. We included a total of 44 patients with M. tuberculosis infection and nine controls. 12 patients had active tuberculosis (8 of them culture-confirmed). The median age was 15 years (range 4.5 to 49). Median urinary neopterin/creatinine ratio in patients with active tuberculosis was 374.1 micromol/mol (129.0 to 1072.3), in patients with latent M. tuberculosis infection it was 142.1 (28.0 to 384.1), and in controls it was 146.0 (40.3 to 200.0), with significantly higher levels in patients with active tuberculosis (p < 0.01). The receiver operating characteristics curve had an area under the curve of 0.84 (95% CI 0.70 to 0.97) (p < 0.01). Conclusions. Urinary neopterin/creatinine ratios are significantly higher in patients with active tuberculosis compared to patients with latent infection and may be a significant predictor of active tuberculosis in patients with M. tuberculosis infection. PMID:27433370

  2. [Increased IL-4 production in response to virulent Mycobacterium tuberculosis in tuberculosis patients with advanced disease].

    PubMed

    Ordway, Diane J; Martins, Marta S; Costa, Leonor M; Freire, Mónica S; Arroz, Maria J; Dockrell, Hazel M; Ventura, Fernando A

    2005-01-01

    The study was designed to compare immune responses to Mycobacterium tuberculosis bacilli and antigens in healthy Portuguese subjects and pulmonary tuberculosis patients (TB), and to correlate immune status with clinical severity of tuberculosis disease. PBMC were cultured and stimulated with live and killed M. tuberculosis H37Rv and purified protein derivative (PPD) and lymphoproliferation and production of IFN-gamma and IL-5/IL-4 by these cultures were evaluated by the use of ELISA and multi-parameter flow cytometry. PBMC from 30 tuberculosis patients demonstrated significantly reduced amounts of proliferation and IFN-gamma when stimulated with live M. tuberculosis compared the control group. Of 15 tuberculosis patients tested for intracellular IL-4 following stimulation with M. tuberculosis, 7 showed greatly increased IL-4 production in CD8+ and gammadelta+ T cells. Tuberculosis patients demonstrated an increase of intracellular IL-4 after PBMC were stimulated with live M. tuberculosis in the CD4+ phenotype, but more notably in CD8+ and gammadelta TCR+ subsets. Increased production of IL-4 in tuberculosis patients was primarily in individuals with advanced involvement of lung parenchymal with high bacterial loads in sputum. These results suggest that an alteration in type 1 and type 2 cytokine balance can occur in patients with tuberculosis at an advanced clinical stage of disease. PMID:16202332

  3. Transmission of Mycobacterium orygis (M. tuberculosis complex species) from a tuberculosis patient to a dairy cow in New Zealand.

    PubMed

    Dawson, Kara L; Bell, Anita; Kawakami, R Pamela; Coley, Kathryn; Yates, Gary; Collins, Desmond M

    2012-09-01

    Mycobacterium orygis, previously called the oryx bacillus, is a member of the Mycobacterium tuberculosis complex and has been reported only recently as a cause of human tuberculosis in patients of South Asian origin. We present the first case documenting the transmission of this organism from a human to a cow. PMID:22785186

  4. Identification of proteins from Mycobacterium tuberculosis missing in attenuated Mycobacterium bovis BCG strains.

    PubMed

    Mattow, J; Jungblut, P R; Schaible, U E; Mollenkopf, H J; Lamer, S; Zimny-Arndt, U; Hagens, K; Müller, E C; Kaufmann, S H

    2001-08-01

    A proteome approach, combining high-resolution two-dimensional electrophoresis (2-DE) with mass spectrometry, was used to compare the cellular protein composition of two virulent strains of Mycobacterium tuberculosis with two attenuated strains of Mycobacterium bovis Bacillus Calmette-Guerin (BCG), in order to identify unique proteins of these strains. Emphasis was given to the identification of M. tuberculosis specific proteins, because we consider these proteins to represent putative virulence factors and interesting candidates for vaccination and diagnosis of tuberculosis. The genome of M. tuberculosis strain H37Rv comprises nearly 4000 predicted open reading frames. In contrast, the separation of proteins from whole mycobacterial cells by 2-DE resulted in silver-stained patterns comprising about 1800 distinct protein spots. Amongst these, 96 spots were exclusively detected either in the virulent (56 spots) or in the attenuated (40 spots) mycobacterial strains. Fifty-three of these spots were analyzed by mass spectrometry, of which 41 were identified, including 32 M. tuberculosis specific spots. Twelve M. tuberculosis specific spots were identified as proteins, encoded by genes previously reported to be deleted in M. bovis BCG. The remaining 20 spots unique for M. tuberculosis were identified as proteins encoded by genes that are not known to be missing in M. bovis BCG. PMID:11565788

  5. Phospholipase C in Beijing strains of Mycobacterium tuberculosis

    PubMed Central

    Mirsamadi, ES; Farnia, P; Jahani Sherafat, S; Esfahani, M; Faramarzi, N

    2010-01-01

    Background and Objectives Phospholipase of Mycobacterium tuberculosis plays an important role in pathogenesis through breaking up phospholipids and production of diacylglycerol. In this study, we examined the Beijing strains of Mycobacterium tuberculosis isolated from Iranian patients for the genes encoding this enzyme. Materials and Methods DNA extraction was performed using CTAB (cetyltrimethylammonium bromide) from positive culture specimens in tuberculosis patients. PCR was then used to amplify the plcA, plcB, plcC genes of Beijing strain, and non-Beijing strains were identified by spoligotyping. Results Of 200 specimens, 19 (9.5%) were Beijing strain and 181 (90.5%) were non-Beijing strains. The results of PCR for Beijing strains were as follows: 16 strains (84.2%) were positive for plcA, 17 (89.4%) were positive for plcB and 17 (89.4%) were positive for plcC genes. The standard strain (H37RV) was used as control. Conclusion The majority of Beijing strains have phospholipase C genes which can contribute to their pathogenesis but we need complementary studies to confirm the role of phospholipase C in pathogenecity of Mycobacterium tuberculosis. PMID:22347572

  6. Identification of Mycobacterium tuberculosis antigens in Seibert fractions by immunoblotting.

    PubMed Central

    Coates, S R; Hansen, D; Schecter, G; Slutkin, G; Hopewell, P; Affronti, L; Echenberg, D F

    1986-01-01

    Seibert fractions prepared from Mycobacterium tuberculosis culture filtrates were evaluated by immunoblotting with a serum pool from patients with active pulmonary tuberculosis. Antibody activity was observed primarily with antigens in the polysaccharide II and A protein fractions; these fractions were further evaluated by immunoblotting with sera from individual patients with tuberculosis, from individuals without tuberculosis and positive for the purified protein derivative antigen skin test, and from individuals negative for the purified protein derivative antigen skin test. The antigens identified in the protein A fraction, a 32,000-molecular-weight antigen and a heterogeneous high-molecular-weight antigen, reacted with antibody found in sera from all patients with tuberculosis and with antibody from over 25% of the control individuals. A 10,000-molecular-weight antigen, a 30,000- to 44,000-molecular-weight antigen, and a heterogeneous high-molecular-weight antigen were observed in the polysaccharide II fraction; these antigens reacted with serum antibody from 70% or more of the patients with tuberculosis and with antibody from 20 to 70% of the control individuals. One of the antigens, with a molecular weight ranging from 17,000 to 28,000 in the polysaccharide II fraction, reacted with antibody in 64% of the sera from patients with tuberculosis but with only 1 of 15 control normal sera. This antigen may elicit an antibody response specifically associated with tuberculosis. Images PMID:3088029

  7. Tuberculosis patients co-infected with Mycobacterium bovis and Mycobacterium tuberculosis in an urban area of Brazil

    PubMed Central

    Silva, Marcio Roberto; Rocha, Adalgiza da Silva; da Costa, Ronaldo Rodrigues; de Alencar, Andrea Padilha; de Oliveira, Vania Maria; Fonseca, Antônio Augusto; Sales, Mariana Lázaro; Issa, Marina de Azevedo; Soares, Paulo Martins; Pereira, Omara Tereza Vianello; dos Santos, Eduardo Calazans; Mendes, Rejane Silva; Ferreira, Ângela Maria de Jesus; Mota, Pedro Moacyr Pinto Coelho; Suffys, Philip Noel; Guimarães, Mark Drew Crosland

    2013-01-01

    In this cross-sectional study, mycobacteria specimens from 189 tuberculosis (TB) patients living in an urban area in Brazil were characterised from 2008-2010 using phenotypic and molecular speciation methods (pncA gene and oxyR pseudogene analysis). Of these samples, 174 isolates simultaneously grew on Löwenstein-Jensen (LJ) and Stonebrink (SB)-containing media and presented phenotypic and molecular profiles of Mycobacterium tuberculosis, whereas 12 had molecular profiles of M. tuberculosis based on the DNA analysis of formalin-fixed paraffin wax-embedded tissue samples (paraffin blocks). One patient produced two sputum isolates, the first of which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, and the second of which only grew on SB media and presented phenotypic profiles of Mycobacterium bovis. One patient provided a bronchial lavage isolate, which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, but had molecular profiles of M. bovis from paraffin block DNA analysis, and one sample had molecular profiles of M. tuberculosis and M. bovis identified from two distinct paraffin blocks. Moreover, we found a low prevalence (1.6%) of M. bovis among these isolates, which suggests that local health service procedures likely underestimate its real frequency and that it deserves more attention from public health officials. PMID:23778657

  8. Evasion and subversion of antigen presentation by Mycobacterium tuberculosis

    PubMed Central

    Baena, Andres; Porcelli, Steven A.

    2009-01-01

    Mycobacterium tuberculosis is one of the most successful of human pathogens, and has acquired the ability to establish latent or progressive infection and persist even in the presence of a fully functioning immune system. The ability of M. tuberculosis to avoid immune-mediated clearance is likely to reflect a highly evolved and coordinated program of immune evasion strategies, including some that interfere with antigen presentation to prevent or alter the quality of T cell responses. Here we review an extensive array of published studies supporting the view that antigen presentation pathways are targeted at many points by pathogenic mycobacteria. These studies reveal the multiple potential mechanisms by which M. tuberculosis may actively inhibit, subvert or otherwise modulate antigen presentation by MHC class I, class II and CD1 molecules. Unraveling the mechanisms by which M. tuberculosis evades or modulates antigen presentation is of critical importance for the development of more effective new vaccines based on live attenuated mycobacterial strains. PMID:19563525

  9. Draft Genome Sequence of a New Zealand Rangipo Strain of Mycobacterium tuberculosis

    PubMed Central

    Gautam, Sanjay S.; Bower, James E.; Basu, Indira

    2016-01-01

    The Rangipo genotype of the Mycobacterium tuberculosis complex has been associated with a number of tuberculosis (TB) outbreaks in New Zealand. We report here the draft whole-genome sequence of a representative isolate of this strain. PMID:27389273

  10. Draft Genome Sequence of Mycobacterium tuberculosis KT-0184, Isolated in South Korea.

    PubMed

    Kwon, Taesoo; Han, Seung Jung; Yoo, Won Gi; Yun, Mi-Ran; Lee, Sanghyun; Lee, Jong Seok; Kim, Dae-Won

    2016-01-01

    Here, we describe the draft genome sequence of Mycobacterium tuberculosis KT-0184, from the Beijing family. This genome will provide insight into the evolution and adaptation of M. tuberculosis KT-0184 in human hosts. PMID:26893431

  11. Draft Genome Sequence of Mycobacterium tuberculosis KT-0204, Isolated in South Korea.

    PubMed

    Yun, Mi-Ran; Han, Seung Jung; Yoo, Won Gi; Kwon, Taesoo; Lee, Sanghyun; Lee, Jong Seok; Kim, Dae-Won

    2016-01-01

    Here, we describe the draft genome sequence of Mycobacterium tuberculosis KT-0204, non-Beijing family. This sequence will reveal genes related to the evolution and adaptation of M. tuberculosis KT-0204 in human hosts. PMID:26847902

  12. Dispersal of Mycobacterium tuberculosis via the Canadian fur trade

    PubMed Central

    Pepperell, Caitlin S.; Granka, Julie M.; Alexander, David C.; Behr, Marcel A.; Chui, Linda; Gordon, Janet; Guthrie, Jennifer L.; Jamieson, Frances B.; Langlois-Klassen, Deanne; Long, Richard; Nguyen, Dao; Wobeser, Wendy; Feldman, Marcus W.

    2011-01-01

    Patterns of gene flow can have marked effects on the evolution of populations. To better understand the migration dynamics of Mycobacterium tuberculosis, we studied genetic data from European M. tuberculosis lineages currently circulating in Aboriginal and French Canadian communities. A single M. tuberculosis lineage, characterized by the DS6Quebec genomic deletion, is at highest frequency among Aboriginal populations in Ontario, Saskatchewan, and Alberta; this bacterial lineage is also dominant among tuberculosis (TB) cases in French Canadians resident in Quebec. Substantial contact between these human populations is limited to a specific historical era (1710–1870), during which individuals from these populations met to barter furs. Statistical analyses of extant M. tuberculosis minisatellite data are consistent with Quebec as a source population for M. tuberculosis gene flow into Aboriginal populations during the fur trade era. Historical and genetic analyses suggest that tiny M. tuberculosis populations persisted for ∼100 y among indigenous populations and subsequently expanded in the late 19th century after environmental changes favoring the pathogen. Our study suggests that spread of TB can occur by two asynchronous processes: (i) dispersal of M. tuberculosis by minimal numbers of human migrants, during which small pathogen populations are sustained by ongoing migration and slow disease dynamics, and (ii) expansion of the M. tuberculosis population facilitated by shifts in host ecology. If generalizable, these migration dynamics can help explain the low DNA sequence diversity observed among isolates of M. tuberculosis and the difficulties in global elimination of tuberculosis, as small, widely dispersed pathogen populations are difficult both to detect and to eradicate. PMID:21464295

  13. Dispersal of Mycobacterium tuberculosis via the Canadian fur trade.

    PubMed

    Pepperell, Caitlin S; Granka, Julie M; Alexander, David C; Behr, Marcel A; Chui, Linda; Gordon, Janet; Guthrie, Jennifer L; Jamieson, Frances B; Langlois-Klassen, Deanne; Long, Richard; Nguyen, Dao; Wobeser, Wendy; Feldman, Marcus W

    2011-04-19

    Patterns of gene flow can have marked effects on the evolution of populations. To better understand the migration dynamics of Mycobacterium tuberculosis, we studied genetic data from European M. tuberculosis lineages currently circulating in Aboriginal and French Canadian communities. A single M. tuberculosis lineage, characterized by the DS6(Quebec) genomic deletion, is at highest frequency among Aboriginal populations in Ontario, Saskatchewan, and Alberta; this bacterial lineage is also dominant among tuberculosis (TB) cases in French Canadians resident in Quebec. Substantial contact between these human populations is limited to a specific historical era (1710-1870), during which individuals from these populations met to barter furs. Statistical analyses of extant M. tuberculosis minisatellite data are consistent with Quebec as a source population for M. tuberculosis gene flow into Aboriginal populations during the fur trade era. Historical and genetic analyses suggest that tiny M. tuberculosis populations persisted for ∼100 y among indigenous populations and subsequently expanded in the late 19th century after environmental changes favoring the pathogen. Our study suggests that spread of TB can occur by two asynchronous processes: (i) dispersal of M. tuberculosis by minimal numbers of human migrants, during which small pathogen populations are sustained by ongoing migration and slow disease dynamics, and (ii) expansion of the M. tuberculosis population facilitated by shifts in host ecology. If generalizable, these migration dynamics can help explain the low DNA sequence diversity observed among isolates of M. tuberculosis and the difficulties in global elimination of tuberculosis, as small, widely dispersed pathogen populations are difficult both to detect and to eradicate. PMID:21464295

  14. Inactivation of Mycobacterium tuberculosis for DNA Typing Analysis

    PubMed Central

    Bemer-Melchior, P.; Drugeon, H. B.

    1999-01-01

    DNA fingerprinting analysis of Mycobacterium tuberculosis is used for epidemiological studies and the control of laboratory cross-contamination. Because standardized procedures are not entirely safe for mycobacteriology laboratory staff, the paper proposes a new technique for the processing of specimens. The technique ensures the inactivation of M. tuberculosis before DNA extraction without the loss of DNA integrity. The control of inactivated cultures should be rigorous and should involve the use of two different culture media incubated for at least 4 months. PMID:10364613

  15. Efficacy of Nitazoxanide against Clinical Isolates of Mycobacterium tuberculosis

    PubMed Central

    Shigyo, Kristina; Ocheretina, Oksana; Merveille, Yves Mary; Johnson, Warren D.; Pape, Jean William; Nathan, Carl F.

    2013-01-01

    Nitazoxanide (NTZ) has bactericidal activity against the H37Rv laboratory strain of Mycobacterium tuberculosis with a MIC of 16 μg/ml. However, its efficacy against clinical isolates of M. tuberculosis has not been determined. We found that NTZ's MIC against 50 clinical isolates ranged from 12 to 28 μg/ml with a median of 16 μg/ml and was unaffected by resistance to first- or second-line antituberculosis drugs or a diversity of spoligotypes. PMID:23507275

  16. Identification of new diamine scaffolds with activity against Mycobacterium tuberculosis

    PubMed Central

    Bogatcheva, Elena; Hanrahan, Colleen; Nikonenko, Boris; Samala, Rowena; Chen, Ping; Gearhart, Jacqueline; Barbosa, Francis; Einck, Leo; Nacy, Carol A.; Protopopova, Marina

    2015-01-01

    A diverse 5,000-compound library was synthesized from commercially available diamines and screened for activity against Mycobacterium tuberculosis in vitro, revealing 143 hits with MIC equal to or less than 12.5 µM. New prospective scaffolds with antitubercular activity derived from homopiperazine, phenyl- and benzyl substituted piperazines, 4-aminomethylpiperidine, 4-aminophenylethylamine, 4,4'-methylenebiscyclohexylamine were identified. Compound SQ775 derived from homopiperazine, and compound SQ786 derived from benzylpiperazine had potent antimicrobial activity against M. tuberculosis in experimental animals in vivo. PMID:16722620

  17. Cholesterol catabolism as a therapeutic target in Mycobacterium tuberculosis

    PubMed Central

    Ouellet, Hugues; Johnston, Jonathan B.; Ortiz de Montellano, Paul R.

    2011-01-01

    Mycobacterium tuberculosis (Mtb) is an intracellular pathogen that infects 10 million worldwide and kills 2 million people every year. The uptake and utilization of nutrients by Mtb within the host cell is still poorly understood, although lipids play an important role in Mtb persistence. The recent identification of a large regulon of cholesterol catabolic genes suggests that Mtb can use host sterol for infection and persistence. In this review, we report on recent progress in elucidation of the Mtb cholesterol catabolic reactions and their potential utility as targets for tuberculosis therapeutic agents. PMID:21924910

  18. Zirconia based nucleic acid sensor for Mycobacterium tuberculosis detection

    NASA Astrophysics Data System (ADS)

    Das, Maumita; Sumana, Gajjala; Nagarajan, R.; Malhotra, B. D.

    2010-03-01

    Nanostructured zirconium oxide (ZrO2) film (particle size˜35 nm), electrochemically deposited onto gold(Au) surface, has been used to immobilize 21-mer oligonucleotide probe (ssDNA) specific to Mycobacterium tuberculosis by utilizing affinity between oxygen atom of phosphoric group and zirconium to fabricate DNA biosensor. This DNA-ZrO2/Au bioelectrode, characterized using x-ray diffraction, Fourier transform infrared spectroscopy, cyclic voltammetry, and scanning electron microscopy techniques, can be used for early and rapid diagnosis of M. tuberculosis with detection limit of 0.065 ng/μL within 60s.

  19. Mycobacterium tuberculosis virulence: insights and impact on vaccine development.

    PubMed

    Delogu, Giovanni; Provvedi, Roberta; Sali, Michela; Manganelli, Riccardo

    2015-01-01

    The existing TB vaccine, the attenuated Mycobacterium bovis strain BCG, is effective in protecting infants from severe forms of the disease, while its efficacy in protecting adults from pulmonary TB is poor. In the last two decades, a renewed interest in TB resulted in the development of several candidate vaccines that are now entering clinical trials. However, most of these vaccines are based on a common rationale and aim to induce a strong T-cell response against Mycobacterium tuberculosis. Recent advancements in the understanding of M. tuberculosis virulence determinants and associated pathogenic strategies are opening a new and broader view of the complex interaction between this remarkable pathogen and the human host, providing insights at molecular level that could lead to a new rationale for the design of novel antitubercular vaccines. A vaccination strategy that simultaneously targets different steps in TB pathogenesis may result in improved protection and reduced TB transmission. PMID:26119086

  20. Complete Genome Sequence of Mycobacterium tuberculosis Clinical Isolate Spoligotype SIT745/EAI1-MYS.

    PubMed

    Suraiya, S; Semail, N; Ismail, M F; Abdullah, J M

    2016-01-01

    Mycobacterium tuberculosis is known to cause pulmonary and extrapulmonary tuberculosis. This organism showed special phylogeographical specificity. Here, we report the complete genome sequence of M. tuberculosis clinical isolate spoligotype SIT745/EAI1-MYS, which was isolated from a Malaysian tuberculosis patient. PMID:27198011

  1. Lymphatic endothelial cells are a replicative niche for Mycobacterium tuberculosis

    PubMed Central

    Lerner, Thomas R.; de Souza Carvalho-Wodarz, Cristiane; Repnik, Urska; Russell, Matthew R.G.; Borel, Sophie; Diedrich, Collin R.; Rohde, Manfred; Wainwright, Helen; Collinson, Lucy M.; Wilkinson, Robert J.; Griffiths, Gareth; Gutierrez, Maximiliano G.

    2016-01-01

    In extrapulmonary tuberculosis, the most common site of infection is within the lymphatic system, and there is growing recognition that lymphatic endothelial cells (LECs) are involved in immune function. Here, we identified LECs, which line the lymphatic vessels, as a niche for Mycobacterium tuberculosis in the lymph nodes of patients with tuberculosis. In cultured primary human LECs (hLECs), we determined that M. tuberculosis replicates both in the cytosol and within autophagosomes, but the bacteria failed to replicate when the virulence locus RD1 was deleted. Activation by IFN-γ induced a cell-autonomous response in hLECs via autophagy and NO production that restricted M. tuberculosis growth. Thus, depending on the activation status of LECs, autophagy can both promote and restrict replication. Together, these findings reveal a previously unrecognized role for hLECs and autophagy in tuberculosis pathogenesis and suggest that hLECs are a potential niche for M. tuberculosis that allows establishment of persistent infection in lymph nodes. PMID:26901813

  2. Lymphatic endothelial cells are a replicative niche for Mycobacterium tuberculosis.

    PubMed

    Lerner, Thomas R; de Souza Carvalho-Wodarz, Cristiane; Repnik, Urska; Russell, Matthew R G; Borel, Sophie; Diedrich, Collin R; Rohde, Manfred; Wainwright, Helen; Collinson, Lucy M; Wilkinson, Robert J; Griffiths, Gareth; Gutierrez, Maximiliano G

    2016-03-01

    In extrapulmonary tuberculosis, the most common site of infection is within the lymphatic system, and there is growing recognition that lymphatic endothelial cells (LECs) are involved in immune function. Here, we identified LECs, which line the lymphatic vessels, as a niche for Mycobacterium tuberculosis in the lymph nodes of patients with tuberculosis. In cultured primary human LECs (hLECs), we determined that M. tuberculosis replicates both in the cytosol and within autophagosomes, but the bacteria failed to replicate when the virulence locus RD1 was deleted. Activation by IFN-γ induced a cell-autonomous response in hLECs via autophagy and NO production that restricted M. tuberculosis growth. Thus, depending on the activation status of LECs, autophagy can both promote and restrict replication. Together, these findings reveal a previously unrecognized role for hLECs and autophagy in tuberculosis pathogenesis and suggest that hLECs are a potential niche for M. tuberculosis that allows establishment of persistent infection in lymph nodes. PMID:26901813

  3. Mycobacterium tuberculosis Infection of Domesticated Asian Elephants, Thailand

    PubMed Central

    Angkawanish, Taweepoke; Sirimalaisuwan, Anucha; Kaewsakhorn, Thattawan; Boonsri, Kittikorn; Rutten, Victor P.M.G.

    2010-01-01

    Four Asian elephants were confirmed to be infected with Mycobacterium tuberculosis by bacterial culture, other diagnostic procedures, and sequencing of 16S–23S rDNA internal transcribed spacer region, 16S rRNA, and gyrase B gene sequences. Genotyping showed that the infectious agents originated from 4 sources in Thailand. To identify infections, a combination of diagnostic assays is essential. PMID:21122228

  4. Mycobacterium tuberculosis Mutations Associated with Reduced Susceptibility to Linezolid.

    PubMed

    Zhang, Shuo; Chen, Jiazhen; Cui, Peng; Shi, Wanliang; Shi, Xiaohong; Niu, Hongxia; Chan, Denise; Yew, Wing Wai; Zhang, Wenhong; Zhang, Ying

    2016-04-01

    Linezolid (LZD) has become increasingly important for the treatment of multidrug-resistant tuberculosis (MDR-TB), but its mechanisms of resistance are not well characterized. We isolated 32 mutants ofMycobacterium tuberculosiswith reduced susceptibility to LZD, which was accounted for byrrlandrplCmutations in almost equal proportions, causing lower and higher MICs, respectively. Our findings provide useful information for the rapid detection of LZD resistance for improved treatment of MDR-TB. PMID:26810645

  5. Carbapenems and Rifampin Exhibit Synergy against Mycobacterium tuberculosis and Mycobacterium abscessus

    PubMed Central

    Kaushik, Amit; Makkar, Nayani; Pandey, Pooja; Parrish, Nicole; Singh, Urvashi

    2015-01-01

    An effective regimen for treatment of tuberculosis (TB) is comprised of multiple drugs that inhibit a range of essential cellular activities in Mycobacterium tuberculosis. The effectiveness of a regimen is further enhanced if constituent drugs act with synergy. Here, we report that faropenem (a penem) or biapenem, doripenem, or meropenem (carbapenems), which belong to the β-lactam class of antibiotics, and rifampin, one of the drugs that forms the backbone of TB treatment, act with synergy when combined. One of the reasons (carba)penems are seldom used for treatment of TB is the high dosage levels required, often at the therapeutic limits. The synergistic combination of rifampin and these (carba)penems indicates that (carba)penems can be administered at dosages that are therapeutically relevant. The combination of faropenem and rifampin also limits the frequency of resistant mutants, as we were unable to obtain spontaneous mutants in the presence of these two drugs. The combinations of rifampin and (carba)penems were effective not only against drug-sensitive Mycobacterium tuberculosis but also against drug-resistant clinical isolates that are otherwise resistant to rifampin. A combination of doripenem or biapenem and rifampin also exhibited synergistic activity against Mycobacterium abscessus. Although the MICs of these three drugs alone against M. abscessus are too high to be of clinical relevance, their concentrations in combinations are therapeutically relevant; therefore, they warrant further evaluation for clinical utility to treat Mycobacterium abscessus infection, especially in cystic fibrosis patients. PMID:26259792

  6. Carbapenems and Rifampin Exhibit Synergy against Mycobacterium tuberculosis and Mycobacterium abscessus.

    PubMed

    Kaushik, Amit; Makkar, Nayani; Pandey, Pooja; Parrish, Nicole; Singh, Urvashi; Lamichhane, Gyanu

    2015-10-01

    An effective regimen for treatment of tuberculosis (TB) is comprised of multiple drugs that inhibit a range of essential cellular activities in Mycobacterium tuberculosis. The effectiveness of a regimen is further enhanced if constituent drugs act with synergy. Here, we report that faropenem (a penem) or biapenem, doripenem, or meropenem (carbapenems), which belong to the β-lactam class of antibiotics, and rifampin, one of the drugs that forms the backbone of TB treatment, act with synergy when combined. One of the reasons (carba)penems are seldom used for treatment of TB is the high dosage levels required, often at the therapeutic limits. The synergistic combination of rifampin and these (carba)penems indicates that (carba)penems can be administered at dosages that are therapeutically relevant. The combination of faropenem and rifampin also limits the frequency of resistant mutants, as we were unable to obtain spontaneous mutants in the presence of these two drugs. The combinations of rifampin and (carba)penems were effective not only against drug-sensitive Mycobacterium tuberculosis but also against drug-resistant clinical isolates that are otherwise resistant to rifampin. A combination of doripenem or biapenem and rifampin also exhibited synergistic activity against Mycobacterium abscessus. Although the MICs of these three drugs alone against M. abscessus are too high to be of clinical relevance, their concentrations in combinations are therapeutically relevant; therefore, they warrant further evaluation for clinical utility to treat Mycobacterium abscessus infection, especially in cystic fibrosis patients. PMID:26259792

  7. Mycobacterium thermoresistibile as a source of thermostable orthologs of Mycobacterium tuberculosis proteins.

    PubMed

    Edwards, Thomas E; Liao, Reiling; Phan, Isabelle; Myler, Peter J; Grundner, Christoph

    2012-07-01

    The genus Mycobacterium comprises major human pathogens such as the causative agent of tuberculosis, Mycobacterium tuberculosis (Mtb), and many environmental species. Tuberculosis claims ~1.5 million lives every year, and drug resistant strains of Mtb are rapidly emerging. To aid the development of new tuberculosis drugs, major efforts are currently under way to determine crystal structures of Mtb drug targets and proteins involved in pathogenicity. However, a major obstacle to obtaining crystal structures is the generation of well-diffracting crystals. Proteins from thermophiles can have better crystallization and diffraction properties than proteins from mesophiles, but their sequences and structures are often divergent. Here, we establish a thermophilic mycobacterial model organism, Mycobacterium thermoresistibile (Mth), for the study of Mtb proteins. Mth tolerates higher temperatures than Mtb or other environmental mycobacteria such as M. smegmatis. Mth proteins are on average more soluble than Mtb proteins, and comparison of the crystal structures of two pairs of orthologous proteins reveals nearly identical folds, indicating that Mth structures provide good surrogates for Mtb structures. This study introduces a thermophile as a source of protein for the study of a closely related human pathogen and marks a new approach to solving challenging mycobacterial protein structures. PMID:22544630

  8. Rapid drug susceptibility test of mycobacterium tuberculosis by bioluminescence sensor

    NASA Astrophysics Data System (ADS)

    Lu, Bin; Xu, Shunqing; Chen, Zifei; Zhou, Yikai

    2001-09-01

    With the persisting increase of drug-resistant stains of M. Tuberculosis around the world, rapid and sensitive detection of antibiotic of M. Tuberculosis is becoming more and more important. In the present study, drug susceptibility of M. tuberculosis were detected by recombination mycobacteriophage combined with bioluminescence sensor. It is based on the use of recombination mycobacteriophage which can express firefly luciferase when it infects viable mycobacteria, and can effectively produce quantifiable photon. Meanwhile, in mycobacterium cells treated with active antibiotic, no light is observed. The emitted light is recorded by a bioluminscence sensor, so the result of drug-resistant test can be determined by the naked eye. 159 stains of M. tuberculosis were applied to this test on their resistant to rifampin, streptomycin and isoniazid. It is found that the agreement of this assay with Liewenstein- Jensen slat is: rifampin 95.60 percent, isoniazid 91.82 percent, streptomycin 88.68 percent, which showed that it is a fast and practical method to scene and detect drug resistant of mycobacterium stains.

  9. Uracil excision repair in Mycobacterium tuberculosis cell-free extracts.

    PubMed

    Kumar, Pradeep; Bharti, Sanjay Kumar; Varshney, Umesh

    2011-05-01

    Uracil excision repair is ubiquitous in all domains of life and initiated by uracil DNA glycosylases (UDGs) which excise the promutagenic base, uracil, from DNA to leave behind an abasic site (AP-site). Repair of the resulting AP-sites requires an AP-endonuclease, a DNA polymerase, and a DNA ligase whose combined activities result in either short-patch or long-patch repair. Mycobacterium tuberculosis, the causative agent of tuberculosis, has an increased risk of accumulating uracils because of its G + C-rich genome, and its niche inside host macrophages where it is exposed to reactive nitrogen and oxygen species, two major causes of cytosine deamination (to uracil) in DNA. In vitro assays to study DNA repair in this important human pathogen are limited. To study uracil excision repair in mycobacteria, we have established assay conditions using cell-free extracts of M. tuberculosis and M. smegmatis (a fast-growing mycobacterium) and oligomer or plasmid DNA substrates. We show that in mycobacteria, uracil excision repair is completed primarily via long-patch repair. In addition, we show that M. tuberculosis UdgB, a newly characterized family 5 UDG, substitutes for the highly conserved family 1 UDG, Ung, thereby suggesting that UdgB might function as backup enzyme for uracil excision repair in mycobacteria. PMID:21371942

  10. The efficacy of the heat killing of Mycobacterium tuberculosis.

    PubMed

    Doig, C; Seagar, A L; Watt, B; Forbes, K J

    2002-10-01

    There is concern that current procedures for the heat inactivation of Mycobacterium tuberculosis may not be adequate. This raises serious safety issues for laboratory staff performing molecular investigations such as IS6110 restriction fragment length polymorphism typing. This paper confirms that the protocol of van Embden et al, as performed routinely in this laboratory, is safe and effective for the heat inactivation of M tuberculosis. This procedure involves complete immersion of a tube containing a suspension of one loopfull of growth in a water bath at 80 degrees C for 20 minutes. Seventy four isolates were included in this investigation. Despite prolonged incubation for 20 weeks, none of the heat killed M tuberculosis suspensions produced visible colonies or gave a positive growth signal from liquid culture. This method did not affect the integrity of the DNA for subsequent molecular investigations. PMID:12354807

  11. Direct identification and typing of Mycobacterium tuberculosis by PCR.

    PubMed Central

    Neimark, H; Ali Baig, M; Carleton, S

    1996-01-01

    We have developed a rapid PCR assay that types strains of Mycobacterium tuberculosis by generating distinct DNA fingerprints directly from primary cultures. This assay allows strain identification analogous to that achieved by the standard restriction fragment length polymorphism method, and fingerprints are obtained in less than 8 h. This assay does not require subculturing, DNA purification, restriction digestion, Southern blotting, or nucleic acid hybridization. Rapid and precise identification of M. tuberculosis strains permits immediate molecular epidemiologic studies. The assay can be converted to a computer-automated system by employing fluorescently labeled PCR primers and the Perkin-Elmer DNA sequencer so that unknown-specimen fingerprints are identified by computer comparison to a database of M. tuberculosis strain fingerprints. PMID:8880499

  12. The efficacy of the heat killing of Mycobacterium tuberculosis

    PubMed Central

    Doig, C; Seagar, A L; Watt, B; Forbes, K J

    2002-01-01

    There is concern that current procedures for the heat inactivation of Mycobacterium tuberculosis may not be adequate. This raises serious safety issues for laboratory staff performing molecular investigations such as IS6110 restriction fragment length polymorphism typing. This paper confirms that the protocol of van Embden et al, as performed routinely in this laboratory, is safe and effective for the heat inactivation of M tuberculosis. This procedure involves complete immersion of a tube containing a suspension of one loopfull of growth in a water bath at 80°C for 20 minutes. Seventy four isolates were included in this investigation. Despite prolonged incubation for 20 weeks, none of the heat killed M tuberculosis suspensions produced visible colonies or gave a positive growth signal from liquid culture. This method did not affect the integrity of the DNA for subsequent molecular investigations. PMID:12354807

  13. Laboratory Diagnosis of Mycobacterium tuberculosis Infection and Disease in Children.

    PubMed

    Dunn, James J; Starke, Jeffrey R; Revell, Paula A

    2016-06-01

    Diagnosis of tuberculosis in children is challenging; even with advanced technologies, the diagnosis is often difficult to confirm microbiologically in part due to the paucibacillary nature of the disease. Clinical diagnosis lacks standardization, and traditional and molecular microbiologic methods lack sensitivity, particularly in children. Immunodiagnostic tests may improve sensitivity, but these tests cannot distinguish tuberculosis disease from latent infection and some lack specificity. While molecular tools like Xpert MTB/RIF have advanced our ability to detect Mycobacterium tuberculosis and to determine antimicrobial resistance, decades old technologies remain the standard in most locales. Today, the battle against this ancient disease still poses one of the primary diagnostic challenges in pediatric laboratory medicine. PMID:26984977

  14. Advances in Mycobacterium tuberculosis therapeutics discovery utlizing structural biology

    PubMed Central

    Chim, Nicholas; Owens, Cedric P.; Contreras, Heidi; Goulding, Celia W.

    2013-01-01

    In 2012, tuberculosis (TB) remains a global health threat and is exacerbated both by the emergence of drug resistant Mycobacterium tuberculosis strains and its synergy with HIV infection. The waning effectiveness of current treatment regimens necessitates the development of new or repurposed anti-TB therapeutics for improved combination therapies against the disease. Exploiting atomic resolution structural information of proteins in complex with their substrates and/or inhibitors can facilitate structure-based rational drug design. Since our last review in 2009, there has been a wealth of new M. tuberculosis protein structural information. Once again, we have compiled the most promising structures with regards to potential anti-TB drug development and present them in this updated review. PMID:23167715

  15. Development of a new DNA extraction protocol for PFGE typing of Mycobacterium tuberculosis complex.

    PubMed

    Ghodousi, Arash; Arash, Ghodousi A; Vatani, S; Darban-Sarokhalil, Davood; Omrani, Maryam; Fooladi, A; Fooladi, Aa; Khosaravi, A; Khosaravi, Ad; Feizabadi, Mohammad Mehdi

    2012-03-01

    A modified pulsed-field gel electrophoresis (PFGE) protocol was developed and applied to clinical isolates of Mycobacterium tuberculosis complex to reduce the cost of using lyticase. This protocol reduces the expense of PFGE typing of Mycobacterium tuberculosis complex as it removes the use of lyticase during the spheroplast formation from these bacteria. PMID:22783461

  16. Development of a new DNA extraction protocol for PFGE typing of Mycobacterium tuberculosis complex

    PubMed Central

    Arash, Ghodousi A; Vatani, S; Darban-Sarokhalil, Davood; Omrani, Maryam; Fooladi, AA; Khosaravi, AD; Feizabadi, Mohammad Mehdi

    2012-01-01

    A modified pulsed-field gel electrophoresis (PFGE) protocol was developed and applied to clinical isolates of Mycobacterium tuberculosis complex to reduce the cost of using lyticase. This protocol reduces the expense of PFGE typing of Mycobacterium tuberculosis complex as it removes the use of lyticase during the spheroplast formation from these bacteria. PMID:22783461

  17. Proteogenomic analysis of Mycobacterium tuberculosis by high resolution mass spectrometry.

    PubMed

    Kelkar, Dhanashree S; Kumar, Dhirendra; Kumar, Praveen; Balakrishnan, Lavanya; Muthusamy, Babylakshmi; Yadav, Amit Kumar; Shrivastava, Priyanka; Marimuthu, Arivusudar; Anand, Sridhar; Sundaram, Hema; Kingsbury, Reena; Harsha, H C; Nair, Bipin; Prasad, T S Keshava; Chauhan, Devendra Singh; Katoch, Kiran; Katoch, Vishwa Mohan; Kumar, Prahlad; Chaerkady, Raghothama; Ramachandran, Srinivasan; Dash, Debasis; Pandey, Akhilesh

    2011-12-01

    The genome sequencing of H37Rv strain of Mycobacterium tuberculosis was completed in 1998 followed by the whole genome sequencing of a clinical isolate, CDC1551 in 2002. Since then, the genomic sequences of a number of other strains have become available making it one of the better studied pathogenic bacterial species at the genomic level. However, annotation of its genome remains challenging because of high GC content and dissimilarity to other model prokaryotes. To this end, we carried out an in-depth proteogenomic analysis of the M. tuberculosis H37Rv strain using Fourier transform mass spectrometry with high resolution at both MS and tandem MS levels. In all, we identified 3176 proteins from Mycobacterium tuberculosis representing ~80% of its total predicted gene count. In addition to protein database search, we carried out a genome database search, which led to identification of ~250 novel peptides. Based on these novel genome search-specific peptides, we discovered 41 novel protein coding genes in the H37Rv genome. Using peptide evidence and alternative gene prediction tools, we also corrected 79 gene models. Finally, mass spectrometric data from N terminus-derived peptides confirmed 727 existing annotations for translational start sites while correcting those for 33 proteins. We report creation of a high confidence set of protein coding regions in Mycobacterium tuberculosis genome obtained by high resolution tandem mass-spectrometry at both precursor and fragment detection steps for the first time. This proteogenomic approach should be generally applicable to other organisms whose genomes have already been sequenced for obtaining a more accurate catalogue of protein-coding genes. PMID:21969609

  18. Granuloma Correlates of Protection Against Tuberculosis and Mechanisms of Immune Modulation by Mycobacterium tuberculosis

    PubMed Central

    Mehra, Smriti; Alvarez, Xavier; Didier, Peter J.; Doyle, Lara A.; Blanchard, James L.; Lackner, Andrew A.; Kaushal, Deepak

    2013-01-01

    Background. The BCG vaccine is ineffective against adult tuberculosis. Hence, new antituberculosis vaccines are needed. Correlates of protection against tuberculosis are not known. We studied the effects of BCG vaccination on gene expression in tuberculosis granulomas using macaques. Methods. Macaques were BCG-vaccinated or sham-vaccinated and then challenged with virulent Mycobacterium tuberculosis. Lung lesions were used for comparative transcriptomics. Results. Vaccinated macaques were protected with lower bacterial burden and immunopathology. Lesions from BCG-vaccinated nonhuman primates (NHPs) showed a better balance of α- and β-chemokine gene expression with higher levels of β-chemokine expression relative to nonvaccinated animals. Consistent with this, sham-vaccinated macaques recruited fewer macrophages relative to neutrophils in their lungs. The expression of indoleamine 2,3-dioxygenase (IDO), a known immunosuppressor, was significantly higher in both week 5 and 10 lesions from sham-vaccinated, relative to BCG-vaccinated, NHPs. IDO expression was primarily limited to the nonlymphocytic region of the lesions, within the inner ring structure surrounding the central necrosis. Conclusions. Our study defines lung gene expression correlates of protective response against tuberculosis, relative to disease, which can potentially be employed to assess the efficacy of candidate antituberculosis vaccines. Mycobacterium tuberculosis may modulate protective immune responses using diverse mechanisms, including increased recruitment of inflammatory neutrophils and the concomitant use of IDO to modulate inflammation. PMID:23255564

  19. Synthetic Long Peptide Derived from Mycobacterium tuberculosis Latency Antigen Rv1733c Protects against Tuberculosis

    PubMed Central

    Coppola, Mariateresa; van den Eeden, Susan J. F.; Wilson, Louis; Franken, Kees L. M. C.; Ottenhoff, Tom H. M.

    2015-01-01

    Responsible for 9 million new cases of active disease and nearly 2 million deaths each year, tuberculosis (TB) remains a global health threat of overwhelming dimensions. Mycobacterium bovis BCG, the only licensed vaccine available, fails to confer lifelong protection and to prevent reactivation of latent infection. Although 15 new vaccine candidates are now in clinical trials, an effective vaccine against TB remains elusive, and new strategies for vaccination are vital. BCG vaccination fails to induce immunity against Mycobacterium tuberculosis latency antigens. Synthetic long peptides (SLPs) combined with adjuvants have been studied mostly for therapeutic cancer vaccines, yet not for TB, and proved to induce efficient antitumor immunity. This study investigated an SLP derived from Rv1733c, a major M. tuberculosis latency antigen which is highly expressed by “dormant” M. tuberculosis and well recognized by T cells from latently M. tuberculosis-infected individuals. In order to assess its in vivo immunogenicity and protective capacity, Rv1733c SLP in CpG was administered to HLA-DR3 transgenic mice. Immunization with Rv1733c SLP elicited gamma interferon-positive/tumor necrosis factor-positive (IFN-γ+/TNF+) and IFN-γ+ CD4+ T cells and Rv1733c-specific antibodies and led to a significant reduction in the bacterial load in the lungs of M. tuberculosis-challenged mice. This was observed both in a pre- and in a post-M. tuberculosis challenge setting. Moreover, Rv1733c SLP immunization significantly boosted the protective efficacy of BCG, demonstrating the potential of M. tuberculosis latency antigens to improve BCG efficacy. These data suggest a promising role for M. tuberculosis latency antigen Rv1733c-derived SLPs as a novel TB vaccine approach, both in a prophylactic and in a postinfection setting. PMID:26202436

  20. Genetic diversity of Mycobacterium tuberculosis isolates from central India

    PubMed Central

    Desikan, Prabha; Chauhan, D.S.; Sharma, Pragya; Panwalkar, Nikita; Chourey, Manju; Patidar, Mohan Lal; Yadav, Priyanka; Chandrasekaran, V.; Ohri, B.S.

    2016-01-01

    Background & objectives: There is a paucity of data available on genetic biodiversity of Mycobacterium tuberculosis isolates from central India. The present study was carried out on isolates of M. tuberculosis cultured from diagnostic clinical samples of patients from Bhopal, central India, using spoligotyping as a method of molecular typing. Methods: DNA was extracted from 340 isolates of M. tuberculosis from culture, confirmed as M. tuberculosis by molecular and biochemical methods and subjected to spoligotyping. The results were compared with the international SITVIT2 database. Results: Sixty five different spoligo international type (SIT) patterns were observed. A total of 239 (70.3%) isolates could be clustered into 25 SITs. The Central Asian (CAS) and East African Indian (EAI) families were found to be the two major circulating families in this region. SIT26/CAS1_DEL was identified as the most predominant type, followed by SIT11/EAI3_IND and SIT288/CAS2. Forty (11.8%) unique (non-clustered) and 61 (17.9%) orphan isolates were identified in the study. There was no significant association of clustering with clinical and demographic characteristics of patients. Interpretation & conclusions: Well established SITs were found to be predominant in our study. SIT26/CAS1_DEL was the most predominant type. However, the occurrence of a substantial number of orphan isolates may indicate the presence of active spatial and temporal evolutionary dynamics within the isolates of M. tuberculosis. PMID:27377505

  1. Bystander Macrophage Apoptosis after Mycobacterium tuberculosis H37Ra Infection▿

    PubMed Central

    Kelly, Deirdre M.; ten Bokum, Annemieke M. C.; O'Leary, Seonadh M.; O'Sullivan, Mary P.; Keane, Joseph

    2008-01-01

    Human macrophages infected with Mycobacterium tuberculosis may undergo apoptosis. Macrophage apoptosis contributes to the innate immune response against M. tuberculosis by containing and limiting the growth of mycobacteria and also by depriving the bacillus of its niche cell. Apoptosis of infected macrophages is well documented; however, bystander apoptosis of uninfected macrophages has not been described in the setting of M. tuberculosis. We observed that uninfected human macrophages underwent significant bystander apoptosis 48 and 96 h after they came into contact with macrophages infected with avirulent M. tuberculosis. The bystander apoptosis was significantly greater than the background apoptosis observed in uninfected control cells cultured for the same length of time. There was no evidence of the involvement of tumor necrosis factor alpha, Fas, tumor necrosis factor-related apoptosis-inducing ligand, transforming growth factor β, Toll-like receptor 2, or MyD88 in contact-mediated bystander apoptosis. This newly described phenomenon may further limit the spread of M. tuberculosis by eliminating the niche cells on which the bacillus relies. PMID:17954721

  2. Structural and functional characterization of Mycobacterium tuberculosis triosephosphate isomerase

    SciTech Connect

    Connor, Sean E.; Capodagli, Glenn C.; Deaton, Michelle K.; Pegan, Scott D.

    2012-04-18

    Tuberculosis (TB) is a major infectious disease that accounts for over 1.7 million deaths every year. Mycobacterium tuberculosis, the causative agent of tuberculosis, enters the human host by the inhalation of infectious aerosols. Additionally, one third of the world's population is likely to be infected with latent TB. The incidence of TB is on the rise owing in part to the emergence of multidrug-resistant strains. As a result, there is a growing need to focus on novel M. tuberculosis enzyme targets. M. tuberculosis triosephosphate isomerase (MtTPI) is an essential enzyme for gluconeogenetic pathways, making it a potential target for future therapeutics. In order to determine its structure, the X-ray crystal structure of MtTPI has been determined, as well as that of MtTPI bound with a reaction-intermediate analog. As a result, two forms of the active site were revealed. In conjunction with the kinetic parameters obtained for the MtTPI-facilitated conversion of dihydroxyacetone phosphate (DHAP) to D-glyceraldehyde-3-phosphate (D-GAP), this provides a greater structural and biochemical understanding of this enzyme. Additionally, isothermal titration calorimetry was used to determine the binding constant for a reaction-intermediate analog bound to the active site of MtTPI.

  3. TIM3 Mediates T Cell Exhaustion during Mycobacterium tuberculosis Infection

    PubMed Central

    Jayaraman, Pushpa; Jacques, Miye K.; Zhu, Chen; Steblenko, Katherine M.; Stowell, Britni L.; Madi, Asaf; Anderson, Ana C.; Kuchroo, Vijay K.; Behar, Samuel M.

    2016-01-01

    While T cell immunity initially limits Mycobacterium tuberculosis infection, why T cell immunity fails to sterilize the infection and allows recrudescence is not clear. One hypothesis is that T cell exhaustion impairs immunity and is detrimental to the outcome of M. tuberculosis infection. Here we provide functional evidence for the development T cell exhaustion during chronic TB. Second, we evaluate the role of the inhibitory receptor T cell immunoglobulin and mucin domain–containing-3 (TIM3) during chronic M. tuberculosis infection. We find that TIM3 expressing T cells accumulate during chronic infection, co-express other inhibitory receptors including PD1, produce less IL-2 and TNF but more IL-10, and are functionally exhausted. Finally, we show that TIM3 blockade restores T cell function and improves bacterial control, particularly in chronically infected susceptible mice. These data show that T cell immunity is suboptimal during chronic M. tuberculosis infection due to T cell exhaustion. Moreover, in chronically infected mice, treatment with anti-TIM3 mAb is an effective therapeutic strategy against tuberculosis. PMID:26967901

  4. Resistance to cellular autophagy by Mycobacterium tuberculosis Beijing strains.

    PubMed

    Haque, Md Fazlul; Boonhok, Rachasak; Prammananan, Therdsak; Chaiprasert, Angkana; Utaisincharoen, Pongsak; Sattabongkot, Jetsumon; Palittapongarnpim, Prasit; Ponpuak, Marisa

    2015-10-01

    Autophagy represents a key pathway in innate immune defense to restrict Mycobacterium tuberculosis growth inside host macrophages. Induction of autophagy has been shown to promote mycobacterial phagosome acidification and acquisition of lysosomal hydrolases, resulting in the elimination of intracellular M. tuberculosis reference strains such as H37Rv. The notorious Beijing genotype has been previously shown to be hyper-virulent and associated with increased survival in host cells and a high mortality rate in animal models, but the underlying mechanism that renders this family to have such advantages remains unclear. We hypothesize that autophagic control against M. tuberculosis Beijing strains may be altered. Here, we discovered that the Beijing strains can resist autophagic killing by host cells compared with that of the reference strain H37Rv and a strain belonging to the East African Indian genotype. Moreover, we have determined a possible underlying mechanism and found that the greater ability to evade autophagic elimination possessed by the Beijing strains stems from their higher capacity to inhibit autophagolysosome biogenesis upon autophagy induction. In summary, a previously unrecognized ability of the M. tuberculosis Beijing strains to evade host autophagy was identified, which may have important implications for tuberculosis treatment, especially in regions prevalent by the Beijing genotype. PMID:26160686

  5. Mycobacterium tuberculosis Pathogenesis and Molecular Determinants of Virulence

    PubMed Central

    Smith, Issar

    2003-01-01

    Tuberculosis (TB), one of the oldest known human diseases. is still is one of the major causes of mortality, since two million people die each year from this malady. TB has many manifestations, affecting bone, the central nervous system, and many other organ systems, but it is primarily a pulmonary disease that is initiated by the deposition of Mycobacterium tuberculosis, contained in aerosol droplets, onto lung alveolar surfaces. From this point, the progression of the disease can have several outcomes, determined largely by the response of the host immune system. The efficacy of this response is affected by intrinsic factors such as the genetics of the immune system as well as extrinsic factors, e.g., insults to the immune system and the nutritional and physiological state of the host. In addition, the pathogen may play a role in disease progression since some M. tuberculosis strains are reportedly more virulent than others, as defined by increased transmissibility as well as being associated with higher morbidity and mortality in infected individuals. Despite the widespread use of an attenuated live vaccine and several antibiotics, there is more TB than ever before, requiring new vaccines and drugs and more specific and rapid diagnostics. Researchers are utilizing information obtained from the complete sequence of the M. tuberculosis genome and from new genetic and physiological methods to identify targets in M. tuberculosis that will aid in the development of these sorely needed antitubercular agents. PMID:12857778

  6. The progress made in determining the Mycobacterium tuberculosis structural proteome

    PubMed Central

    Hecker, Michael

    2011-01-01

    Mycobacterium tuberculosis is a highly infectious pathogen that is still responsible for millions of deaths annually. Effectively treating this disease typically requires a course of antibiotics, most of which were developed decades ago. These drugs are, however, not effective against persistent tubercle bacilli and the emergence of drug-resistant stains threatens to make many of them obsolete. The identification of new drug targets, allowing the development of new potential drugs, is therefore imperative. Both proteomics and structural biology have important roles to play in this process, the former as a means of identifying promising drug targets and the latter allowing understanding of protein function and protein–drug interactions at atomic resolution. The determination of M. tuberculosis protein structures has been a goal of the scientific community for the last decade, who have aimed to supply a large amount of structural data that can be used in structure-based approaches for drug discovery and design. Only since the genome sequence of M. tuberculosis has been available has the determination of large numbers of tuberculosis protein structures been possible. Currently, the molecular structures of 8.5% of all the pathogen's protein-encoding ORFs have been determined. In this review, we look at the progress made in determining the M. tuberculosis structural proteome and the impact this has had on the development of potential new drugs, as well as the discovery of the function of crucial mycobaterial proteins. PMID:21674801

  7. Gene Transfer in Mycobacterium tuberculosis: Shuttle Phasmids to Enlightenment

    PubMed Central

    JACOBS, WILLIAM R.

    2016-01-01

    Infectious diseases have plagued humankind throughout history and have posed serious public health problems. Yet vaccines have eradicated smallpox and antibiotics have drastically decreased the mortality rate of many infectious agents. These remarkable successes in the control of infections came from knowing the causative agents of the diseases, followed by serendipitous discoveries of attenuated viruses and antibiotics. The discovery of DNA as genetic material and the understanding of how this information translates into specific phenotypes have changed the paradigm for developing new vaccines, drugs, and diagnostic tests. Knowledge of the mechanisms of immunity and mechanisms of action of drugs has led to new vaccines and new antimicrobial agents. The key to the acquisition of the knowledge of these mechanisms has been identifying the elemental causes (i.e., genes and their products) that mediate immunity and drug resistance. The identification of these genes is made possible by being able to transfer the genes or mutated forms of the genes into causative agents or surrogate hosts. Such an approach was limited in Mycobacterium tuberculosis by the difficulty of transferring genes or alleles into M. tuberculosis or a suitable surrogate mycobacterial host. The construction of shuttle phasmids—chimeric molecules that replicate in Escherichia coli as plasmids and in mycobacteria as mycobacteriophages—was instrumental in developing gene transfer systems for M. tuberculosis. This review will discuss M. tuberculosis genetic systems and their impact on tuberculosis research. “I had to know my enemy in order to prevail against him.”Nelson Mandela PMID:26105819

  8. Mycobacterium tuberculosis infection in women with unexplained infertility

    PubMed Central

    Eftekhar, Maryam; Pourmasumi, Soheila; Sabeti, Parvin; Aflatoonian, Abbas; Sheikhha, Mohammad Hasan

    2015-01-01

    Background: Genital tuberculosis (GTB) is an important cause of female infertility, especially in developing countries. The positive results of polymerase chain reaction (PCR) in endometrial GTB in the absence of tubal damage raise the possibility of the detection of sub-clinical or latent disease, with doubtful benefits of treatment. Objective: To evaluate the mycobacterium tuberculosis infection in endometrial biopsy samples collected from unexplained infertile women attending Yazd Research and Clinical Center for Infertility by using PCR techniques. Materials and Methods: In this cross sectional study, 144 infertile women with unexplained infertility aged 20-35 years old and normal Histro-saplango graphy findings were enrolled. Endometrial biopsy samples from each participant were tested for mycobacterium tuberculosis detecting by PCR. In 93 patients, peritoneal fluid was also taken for culture and PCR. Results: The PCR results of endometrial specimens were negative in all cases, demonstrating that there was no GTB infection among our patients. Conclusion: Our results showed that GTB could not be considered as a major problem in women with unexplained infertility. Although, studies have indicated that PCR is a useful method in diagnosing early GTB disease in infertile women with no demonstrable evidence of tubal or endometrial involvement. PMID:27141534

  9. Dielectrophoretic characterization of antibiotic-treated Mycobacterium tuberculosis complex cells.

    PubMed

    Inoue, Shinnosuke; Lee, Hyun-Boo; Becker, Annie L; Weigel, Kris M; Kim, Jong-Hoon; Lee, Kyong-Hoon; Cangelosi, Gerard A; Chung, Jae-Hyun

    2015-10-01

    Multi-drug resistant tuberculosis (MDR-TB) has become a serious concern for proper treatment of patients. As a phenotypic method, dielectrophoresis can be useful but is yet to be attempted to evaluate Mycobacterium tuberculosis complex cells. This paper investigates the dielectrophoretic behavior of Mycobacterium bovis (Bacillus Calmette-Guérin, BCG) cells that are treated with heat or antibiotics rifampin (RIF) or isoniazid (INH). The experimental parameters are designed on the basis of our sensitivity analysis. The medium conductivity (σ(m)) and the frequency (f) for a crossover frequency (f(xo1)) test are decided to detect the change of σ(m)-f(xo1) in conjunction with the drug mechanism. Statistical modeling is conducted to estimate the distributions of viable and nonviable cells from the discrete measurement of f (xo1). Finally, the parameters of the electrophysiology of BCG cells, C(envelope) and σ(cyto), are extracted through a sampling algorithm. This is the first evaluation of the dielectrophoresis (DEP) approach as a means to assess the effects of antimicrobial drugs on M. tuberculosis complex cells. PMID:26231690

  10. Structural and Biochemical Characterization of Mycobacterium tuberculosis CYP142

    PubMed Central

    Driscoll, Max D.; McLean, Kirsty J.; Levy, Colin; Mast, Natalia; Pikuleva, Irina A.; Lafite, Pierre; Rigby, Stephen E. J.; Leys, David; Munro, Andrew W.

    2010-01-01

    The Mycobacterium tuberculosis cytochrome P450 enzyme CYP142 is encoded in a large gene cluster involved in metabolism of host cholesterol. CYP142 was expressed and purified as a soluble, low spin P450 hemoprotein. CYP142 binds tightly to cholesterol and its oxidized derivative cholest-4-en-3-one, with extensive shift of the heme iron to the high spin state. High affinity for azole antibiotics was demonstrated, highlighting their therapeutic potential. CYP142 catalyzes either 27-hydroxylation of cholesterol/cholest-4-en-3-one or generates 5-cholestenoic acid/cholest-4-en-3-one-27-oic acid from these substrates by successive sterol oxidations, with the catalytic outcome dependent on the redox partner system used. The CYP142 crystal structure was solved to 1.6 Å, revealing a similar active site organization to the cholesterol-metabolizing M. tuberculosis CYP125, but having a near-identical organization of distal pocket residues to the branched fatty acid oxidizing M. tuberculosis CYP124. The cholesterol oxidizing activity of CYP142 provides an explanation for previous findings that ΔCYP125 strains of Mycobacterium bovis and M. bovis BCG cannot grow on cholesterol, because these strains have a defective CYP142 gene. CYP142 is revealed as a cholesterol 27-oxidase with likely roles in host response modulation and cholesterol metabolism. PMID:20889498

  11. Overview and phylogeny of Mycobacterium tuberculosis complex organisms: implications for diagnostics and legislation of bovine tuberculosis.

    PubMed

    Rodriguez-Campos, Sabrina; Smith, Noel H; Boniotti, Maria B; Aranaz, Alicia

    2014-10-01

    Members of the Mycobacterium tuberculosis complex (MTBC) cause a serious disease with similar pathology, tuberculosis; in this review, bovine tuberculosis will be considered as disease caused by any member of the MTBC in bovids. Bovine tuberculosis is responsible for significant economic loss due to costly eradication programs and trade limitations and poses a threat to both endangered and protected species as well as to public health. We here give an overview on all members of the MTBC, focusing on their isolation from different animal hosts. We also review the recent advances made in elucidating the evolutionary and phylogenetic relationships of members of the MTBC. Because the nomenclature of the MTBC is controversial, its members have been considered species, subspecies or ecotypes, this review discusses the possible implications for diagnostics and the legal consequences of naming of new species. PMID:24630673

  12. Bacterial Proteasome Activator Bpa (Rv3780) Is a Novel Ring-Shaped Interactor of the Mycobacterial Proteasome

    PubMed Central

    Delley, Cyrille L.; Laederach, Juerg; Ziemski, Michal; Bolten, Marcel; Boehringer, Daniel; Weber-Ban, Eilika

    2014-01-01

    The occurrence of the proteasome in bacteria is limited to the phylum of actinobacteria, where it is maintained in parallel to the usual bacterial compartmentalizing proteases. The role it plays in these organisms is still not fully understood, but in the human pathogen Mycobacterium tuberculosis (Mtb) the proteasome supports persistence in the host. In complex with the ring-shaped ATPase Mpa (called ARC in other actinobacteria), the proteasome can degrade proteins that have been post-translationally modified with the prokaryotic ubiquitin-like protein Pup. Unlike for the eukaryotic proteasome core particle, no other bacterial proteasome interactors have been identified to date. Here we describe and characterize a novel bacterial proteasome activator of Mycobacterium tuberculosis we termed Bpa (Rv3780), using a combination of biochemical and biophysical methods. Bpa features a canonical C-terminal proteasome interaction motif referred to as the HbYX motif, and its orthologs are only found in those actinobacteria encoding the proteasomal subunits. Bpa can inhibit degradation of Pup-tagged substrates in vitro by competing with Mpa for association with the proteasome. Using negative-stain electron microscopy, we show that Bpa forms a ring-shaped homooligomer that can bind coaxially to the face of the proteasome cylinder. Interestingly, Bpa can stimulate the proteasomal degradation of the model substrate β-casein, which suggests it could play a role in the removal of non-native or damaged proteins. PMID:25469515

  13. Interaction of Mycobacterium tuberculosis with Host Cell Death Pathways

    PubMed Central

    Srinivasan, Lalitha; Ahlbrand, Sarah; Briken, Volker

    2014-01-01

    Mycobacterium tuberculosis (Mtb) has coevolved with humans for tens of thousands of years. It is thus highly adapted to its human host and has evolved multiple mechanisms to manipulate host immune responses to its advantage. One central host pathogen interaction modality is host cell death pathways. Host cell apoptosis is associated with a protective response to Mtb infection, whereas a necrotic response favors the pathogen. Consistently, Mtb inhibits host cell apoptosis signaling but promotes induction of programmed necrosis. The molecular mechanisms involved in Mtb-mediated host cell death manipulation, the consequences for host immunity, and the potential for therapeutic and preventive approaches will be discussed. PMID:24968864

  14. Anti-Mycobacterium tuberculosis activity of fungus Phomopsis stipata

    PubMed Central

    de Prince, Karina Andrade; Sordi, Renata; Pavan, Fernando Rogério; Barreto Santos, Adolfo Carlos; Araujo, Angela R.; Leite, Sergio R.A.; Leite, Clarice Q. F.

    2012-01-01

    Our purpose was to determine the anti-Mycobacterium tuberculosis activity of the metabolites produced by the endophitic fungus Phomopsis stipata (Lib.) B. Sutton, (Diaporthaceae), cultivated in different media. The antimycobacterial activity was assessed through the Resazurin Microtiter Assay (REMA) and the cytotoxicity test performed on macrophage cell line. The extracts derived from fungi grown on Corn Medium and Potato Dextrose Broth presented the smallest values of Minimum Inhibitory Concentration (MIC) and low cytotoxicity, which implies a high selectivity index. This is the first report on the chemical composition and antitubercular activity of metabolites of P. stipata, as well as the influence of culture medium on these properties. PMID:24031821

  15. Autolysis and Secondary Growth of Mycobacterium tuberculosis in Submerged Culture

    PubMed Central

    Wayne, Lawrence G.; Diaz, Gilbert A.

    1967-01-01

    Mycobacterium tuberculosis has been found to exhibit autolysis and spontaneous secondary growth in modified Sauton medium. The phenomenon is a function of high glycerol concentration of the medium and limitation of air in test tubes. It is not a function of depletion of nutrients and is probably not a manifestation of lysogeny or spheroplast formation. The cycle of growth, autolysis, and secondary growth is related to development of lethal conditions during a period of severe oxygen limitation followed by a metabolic change associated with slower depletion of glycerol. PMID:4962059

  16. Genetic regulation of vesiculogenesis and immunomodulation in Mycobacterium tuberculosis

    PubMed Central

    Rath, Poonam; Huang, Chengdong; Wang, Tao; Wang, Tianzhi; Li, Huilin; Prados-Rosales, Rafael; Elemento, Olivier; Casadevall, Arturo; Nathan, Carl F.

    2013-01-01

    Mycobacterium tuberculosis (Mtb) restrains immune responses well enough to escape eradication but elicits enough immunopathology to ensure its transmission. Here we provide evidence that this host–pathogen relationship is regulated in part by a cytosolic, membrane-associated protein with a unique structural fold, encoded by the Mtb gene rv0431. The protein acts by regulating the quantity of Mtb-derived membrane vesicles bearing Toll-like receptor 2 ligands, including the lipoproteins LpqH and SodC. We propose that rv0431 be named “vesiculogenesis and immune response regulator.” PMID:24248369

  17. Zoonotic tuberculosis due to Mycobacterium bovis in developing countries.

    PubMed Central

    Cosivi, O.; Grange, J. M.; Daborn, C. J.; Raviglione, M. C.; Fujikura, T.; Cousins, D.; Robinson, R. A.; Huchzermeyer, H. F.; de Kantor, I.; Meslin, F. X.

    1998-01-01

    The World Health Organization (WHO) estimates that human tuberculosis (TB) incidence and deaths for 1990 to 1999 will be 88 million and 30 million, respectively, with most cases in developing countries. Zoonotic TB (caused by Mycobacterium bovis) is present in animals in most developing countries where surveillance and control activities are often inadequate or unavailable; therefore, many epidemiologic and public health aspects of infection remain largely unknown. We review available information on zoonotic TB in developing countries, analyze risk factors that may play a role in the disease, review recent WHO activities, and recommend actions to assess the magnitude of the problem and control the disease in humans and animals. PMID:9452399

  18. Antimicrobial Resistance in Mycobacterium tuberculosis: The Odd One Out.

    PubMed

    Eldholm, Vegard; Balloux, François

    2016-08-01

    Antimicrobial resistance (AMR) threats are typically represented by bacteria capable of extensive horizontal gene transfer (HGT). One clear exception is Mycobacterium tuberculosis (Mtb). It is an obligate human pathogen with limited genetic diversity and a low mutation rate which lacks any evidence for HGT. Such features should, in principle, reduce its ability to rapidly evolve AMR. We identify key features in its biology and epidemiology that allow it to overcome its low adaptive potential. We focus in particular on its innate resistance to drugs, its unusual life cycle, including an often extensive latent phase, and its ability to shelter from exposure to antimicrobial drugs within cavities it induces in the lungs. PMID:27068531

  19. Mycobacterium terrae: a potential surrogate for Mycobacterium tuberculosis in a standard disinfectant test.

    PubMed

    Griffiths, P A; Babb, J R; Fraise, A P

    1998-03-01

    The susceptibility of Mycobacterium tuberculosis and Mycobacterium avium-intracellulare to the disinfections used for spillage and heat sensitive instruments has received much attention in recent years. The use of clinical isolates of M. tuberculosis and M. avium-intracellulare as test organisms is considered unsuitable for standard tests due to their hazardous nature (category 3 pathogens and slow growth rates). This has led to much debate in standards committees on the selection and use of a possible surrogate which would be safer and more practical to use and yet mimic the susceptibility of clinical isolates. This study compared the susceptibility of one possible surrogate Mycobacterium terrae NCTC 10856, with that of clinical isolates of M. tuberculosis H37 Rv and M. avium-intracellulare using a quantitative suspension test. The instrument and environmental disinfectants tested were a chlorine-releasing agent, sodium dichloroisocyanyurate (NaDCC) at 1000 ppm and 10,000 ppm av. Cl, chlorine dioxide at 1100 ppm av. ClO2 (Tristel, HayMan MediChem), 0.35% peracetic acid (NuCidex, Johnson & Johnson), 70% industrial methylated spirit (IMS), 2% alkaline glutaraldehyde (Asep, Galen), 10% succine dialdehyde and formaldehyde mixture (Gigasept, Schulke and Mayr). Results showed that the clinical isolate of M. avium-intracellulare was the most resistant of the three test organisms. M. terrae, which is not a category 3 pathogen, was slightly more resistant than M. tuberculosis and this would appear to be a suitable surrogate for establishing tuberculocidal activity. However, with an increase in the clinical significance of M. avium-intracellulare, particularly in human immunodeficiency virus (HIV) and immunocompromised patients, a more resistant surrogate is required. In the absence of such a surrogate, testing with M. avium-intracellulare in a clinical laboratory equipped for handling category 3 pathogens is still advised to establish mycobactericidal activity. PMID

  20. Mycobacterium tuberculosis Infection following Kidney Transplantation

    PubMed Central

    Boubaker, Karima; Gargah, Tahar; Abderrahim, Ezzedine; Ben Abdallah, Taieb; Kheder, Adel

    2013-01-01

    Introduction and Aims. Post-transplant tuberculosis (TB) is a problem in successful long-term outcome of renal transplantation recipients. Our objective was to describe the pattern and risk factors of TB infection and the prognosis in our transplant recipients. Patients and Methods. This study was a retrospective review of the records of 491 renal transplant recipients in our hospital during the period from January 1986 to December 2009. The demographic data, transplant characteristics, clinical manifestations, diagnostic criteria, treatment protocol, and long-term outcome of this cohort of patients were analyzed. Results. 16 patients (3,2%) developed post-transplant TB with a mean age of 32,5 ± 12,7 (range: 13–60) years and a mean post-transplant period of 36,6months (range: 12,3 months–15,9 years). The forms of the diseases were pulmonary in 10/16 (62,6%), disseminated in 3/16 (18,7%), and extrapulmonary in 3/16 (18,7%). Graft dysfunction was observed in 7 cases (43,7%) with tissue-proof acute rejection in 3 cases and loss of the graft in 4 cases. Hepatotoxicity developed in 3 patients (18,7%) during treatment. Recurrences were observed in 4 cases after early stop of treatment. Two patients (12.5%) died. Conclusion. Extra pulmonary and disseminated tuberculosis were observed in third of our patients. More than 9months of treatment may be necessary to prevent recurrence. PMID:24222903

  1. The transmission of Mycobacterium tuberculosis in high burden settings.

    PubMed

    Yates, Tom A; Khan, Palwasha Y; Knight, Gwenan M; Taylor, Jonathon G; McHugh, Timothy D; Lipman, Marc; White, Richard G; Cohen, Ted; Cobelens, Frank G; Wood, Robin; Moore, David A J; Abubakar, Ibrahim

    2016-02-01

    Unacceptable levels of Mycobacterium tuberculosis transmission are noted in high burden settings and a renewed focus on reducing person-to-person transmission in these communities is needed. We review recent developments in the understanding of airborne transmission. We outline approaches to measure transmission in populations and trials and describe the Wells-Riley equation, which is used to estimate transmission risk in indoor spaces. Present research priorities include the identification of effective strategies for tuberculosis infection control, improved understanding of where transmission occurs and the transmissibility of drug-resistant strains, and estimates of the effect of HIV and antiretroviral therapy on transmission dynamics. When research is planned and interventions are designed to interrupt transmission, resource constraints that are common in high burden settings-including shortages of health-care workers-must be considered. PMID:26867464

  2. Microfold Cells Actively Translocate Mycobacterium tuberculosis to Initiate Infection.

    PubMed

    Nair, Vidhya R; Franco, Luis H; Zacharia, Vineetha M; Khan, Haaris S; Stamm, Chelsea E; You, Wu; Marciano, Denise K; Yagita, Hideo; Levine, Beth; Shiloh, Michael U

    2016-08-01

    The prevailing paradigm is that tuberculosis infection is initiated when patrolling alveolar macrophages and dendritic cells within the terminal alveolus ingest inhaled Mycobacterium tuberculosis (Mtb). However, definitive data for this model are lacking. Among the epithelial cells of the upper airway, a specialized epithelial cell known as a microfold cell (M cell) overlies various components of mucosa-associated lymphatic tissue. Here, using multiple mouse models, we show that Mtb invades via M cells to initiate infection. Intranasal Mtb infection in mice lacking M cells either genetically or by antibody depletion resulted in reduced invasion and dissemination to draining lymph nodes. M cell-depleted mice infected via aerosol also had delayed dissemination to lymph nodes and reduced mortality. Translocation of Mtb across two M cell transwell models was rapid and transcellular. Thus, M cell translocation is a vital entry mechanism that contributes to the pathogenesis of Mtb. PMID:27452467

  3. Selective Mycobacterium tuberculosis Shikimate Kinase Inhibitors as Potential Antibacterials

    PubMed Central

    Gordon, Sara; Simithy, Johayra; Goodwin, Douglas C; Calderón, Angela I

    2015-01-01

    Owing to the persistence of tuberculosis (TB) as well as the emergence of multidrug-resistant and extensively drug-resistant (XDR) forms of the disease, the development of new antitubercular drugs is crucial. Developing inhibitors of shikimate kinase (SK) in the shikimate pathway will provide a selective target for antitubercular agents. Many studies have used in silico technology to identify compounds that are anticipated to interact with and inhibit SK. To a much more limited extent, SK inhibition has been evaluated by in vitro methods with purified enzyme. Currently, there are no data on in vivo activity of Mycobacterium tuberculosis shikimate kinase (MtSK) inhibitors available in the literature. In this review, we present a summary of the progress of SK inhibitor discovery and evaluation with particular attention toward development of new antitubercular agents. PMID:25861218

  4. A novel peptide interferes with Mycobacterium tuberculosis virulence and survival

    PubMed Central

    Samuchiwal, Sachin Kumar; Tousif, Sultan; Singh, Dhiraj Kumar; Kumar, Arun; Ghosh, Anamika; Bhalla, Kuhulika; Prakash, Prem; Kumar, Sushil; Trivedi, Ashish Chandra; Bhattacharyya, Maitree; Das, Gobardhan; Ranganathan, Anand

    2014-01-01

    Tuberculosis (TB) is a huge global burden, with new and resistant strains emerging at an alarming rate, necessitating an urgent need for a new class of drug candidates. Here, we report that SL3, a novel 33-amino acid peptide, causes debilitating effects on mycobacterial morphology. Treatment with SL3 drastically inhibits the growth of Mycobacterium tuberculosis in vitro as well as in a pre-clinical mouse model for M.tb infection. Microarray analysis of SL3-expressing strain demonstrates wide-scale transcriptional disruption in M.tb. We therefore believe that SL3 and similar peptides may herald a new approach towards discovering new molecules for TB therapy. PMID:25349777

  5. Benzothiazinones Kill Mycobacterium tuberculosis by Blocking Arabinan Synthesis

    PubMed Central

    Makarov, Vadim; Manina, Giulia; Mikusova, Katarina; Möllmann, Ute; Ryabova, Olga; Saint-Joanis, Brigitte; Dhar, Neeraj; Pasca, Maria Rosalia; Buroni, Silvia; Lucarelli, Anna Paola; Milano, Anna; De Rossi, Edda; Belanova, Martina; Bobovska, Adela; Dianiskova, Petronela; Kordulakova, Jana; Sala, Claudia; Fullam, Elizabeth; Schneider, Patricia; McKinney, John D.; Brodin, Priscille; Christophe, Thierry; Waddell, Simon; Butcher, Philip; Albrethsen, Jakob; Rosenkrands, Ida; Brosch, Roland; Nandi, Vrinda; Bharath, Sowmya; Gaonkar, Sheshagiri; Shandil, Radha K.; Balasubramanian, Venkataraman; Balganesh, Tanjore; Tyagi, Sandeep; Grosset, Jacques; Riccardi, Giovanna; Cole, Stewart T.

    2011-01-01

    New drugs are required to counter the tuberculosis (TB) pandemic. Here, we describe the synthesis and characterization of 1,3-benzothiazin-4-ones (BTZs), a new class of antimycobacterial agents that kill Mycobacterium tuberculosis in vitro, ex vivo, and in mouse models of TB. Using genetics and biochemistry, we identified the enzyme decaprenylphosphoryl-β-d-ribose 2′-epimerase as a major BTZ target. Inhibition of this enzymatic activity abolishes the formation of decaprenylphosphoryl arabinose, a key precursor that is required for the synthesis of the cell-wall arabinans, thus provoking cell lysis and bacterial death. The most advanced compound, BTZ043, is a candidate for inclusion in combination therapies for both drug-sensitive and extensively drug-resistant TB. PMID:19299584

  6. Benzothiazinones kill Mycobacterium tuberculosis by blocking arabinan synthesis.

    PubMed

    Makarov, Vadim; Manina, Giulia; Mikusova, Katarina; Möllmann, Ute; Ryabova, Olga; Saint-Joanis, Brigitte; Dhar, Neeraj; Pasca, Maria Rosalia; Buroni, Silvia; Lucarelli, Anna Paola; Milano, Anna; De Rossi, Edda; Belanova, Martina; Bobovska, Adela; Dianiskova, Petronela; Kordulakova, Jana; Sala, Claudia; Fullam, Elizabeth; Schneider, Patricia; McKinney, John D; Brodin, Priscille; Christophe, Thierry; Waddell, Simon; Butcher, Philip; Albrethsen, Jakob; Rosenkrands, Ida; Brosch, Roland; Nandi, Vrinda; Bharath, Sowmya; Gaonkar, Sheshagiri; Shandil, Radha K; Balasubramanian, Venkataraman; Balganesh, Tanjore; Tyagi, Sandeep; Grosset, Jacques; Riccardi, Giovanna; Cole, Stewart T

    2009-05-01

    New drugs are required to counter the tuberculosis (TB) pandemic. Here, we describe the synthesis and characterization of 1,3-benzothiazin-4-ones (BTZs), a new class of antimycobacterial agents that kill Mycobacterium tuberculosis in vitro, ex vivo, and in mouse models of TB. Using genetics and biochemistry, we identified the enzyme decaprenylphosphoryl-beta-d-ribose 2'-epimerase as a major BTZ target. Inhibition of this enzymatic activity abolishes the formation of decaprenylphosphoryl arabinose, a key precursor that is required for the synthesis of the cell-wall arabinans, thus provoking cell lysis and bacterial death. The most advanced compound, BTZ043, is a candidate for inclusion in combination therapies for both drug-sensitive and extensively drug-resistant TB. PMID:19299584

  7. Iron Acquisition Mechanisms: Promising Target Against Mycobacterium tuberculosis

    PubMed Central

    Hameed, Saif; Pal, Rahul; Fatima, Zeeshan

    2015-01-01

    Continuous deployment of antitubercular drugs in treating Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) has led to the emergence of drug resistance resulting in cross-resistance to many unrelated drugs, a phenomenon termed as Multi-Drug Resistance (MDR-TB). Despite reasonable documentation of major factors which contribute to MDR mechanisms, it appears unavoidable to consider novel mechanisms combating MDR. The ability of pathogenic MTB, to sense and become accustomed to changes in the host environment is essential for its survival and confers the basis of their success as dreadful pathogen. One such significant environmental factor that MTB must surmount is iron limitation, since they encounter diverse anatomical sites during the establishment of infection within the host. Considering the importance of MTB, being the second most common cause of mortality, this review focuses on gaining insights of iron acquisition mechanisms in MTB and how it can be exploited as efficient anti-mycobacterial drug target. PMID:26464608

  8. Progress in targeting cell envelope biogenesis in Mycobacterium tuberculosis

    PubMed Central

    Jackson, Mary; McNeil, Michael R; Brennan, Patrick J

    2013-01-01

    Most of the newly discovered compounds showing promise for the treatment of TB, notably multidrug-resistant TB, inhibit aspects of Mycobacterium tuberculosis cell envelope metabolism. This review reflects on the evolution of the knowledge that many of the front-line and emerging products inhibit aspects of cell envelope metabolism and in the process are bactericidal not only against actively replicating M. tuberculosis, but contrary to earlier impressions, are effective against latent forms of the disease. While mycolic acid and arabinogalactan synthesis are still primary targets of existing and new drugs, peptidoglycan synthesis, transport mechanisms and the synthesis of the decaprenyl-phosphate carrier lipid all show considerable promise as targets for new products, older drugs and new combinations. The advantages of whole cell- versus target-based screening in the perpetual search for new targets and products to counter multidrug-resistant TB are discussed. PMID:23841633

  9. Mycobacterium tuberculosis infection of the 'non-classical immune cell'.

    PubMed

    Randall, Philippa J; Hsu, Nai-Jen; Quesniaux, Valerie; Ryffel, Bernhard; Jacobs, Muazzam

    2015-10-01

    Mycobacterium tuberculosis can infect 'non-classical immune cells', which comprise a significant constituency of cells that reside outside of those defined as 'classical immune cells' from myeloid or lymphoid origin. Here we address the influence of specific 'non-classical immune cells' in host responses and their effects in controlling mycobacterial growth or enabling an environment conducive for bacilli persistence. The interaction of M. tuberculosis with epithelial cells, endothelial cells, fibroblasts, adipocytes, glia and neurons and downstream cellular responses that often dictate immune regulation and disease outcome are discussed. Functional integration and synergy between 'classical' and 'non-classical immune cells' are highlighted as critical for determining optimal immune outcomes that favour the host. PMID:25801479

  10. Selective Mycobacterium tuberculosis Shikimate Kinase Inhibitors as Potential Antibacterials.

    PubMed

    Gordon, Sara; Simithy, Johayra; Goodwin, Douglas C; Calderón, Angela I

    2015-01-01

    Owing to the persistence of tuberculosis (TB) as well as the emergence of multidrug-resistant and extensively drug-resistant (XDR) forms of the disease, the development of new antitubercular drugs is crucial. Developing inhibitors of shikimate kinase (SK) in the shikimate pathway will provide a selective target for antitubercular agents. Many studies have used in silico technology to identify compounds that are anticipated to interact with and inhibit SK. To a much more limited extent, SK inhibition has been evaluated by in vitro methods with purified enzyme. Currently, there are no data on in vivo activity of Mycobacterium tuberculosis shikimate kinase (MtSK) inhibitors available in the literature. In this review, we present a summary of the progress of SK inhibitor discovery and evaluation with particular attention toward development of new antitubercular agents. PMID:25861218

  11. Proposing Low-Similarity Peptide Vaccines against Mycobacterium tuberculosis

    PubMed Central

    Lucchese, Guglielmo; Stufano, Angela; Kanduc, Darja

    2010-01-01

    Using the currently available proteome databases and based on the concept that a rare sequence is a potential epitope, epitopic sequences derived from Mycobacterium tuberculosis were examined for similarity score to the proteins of the host in which the epitopes were defined. We found that: (i) most of the bacterial linear determinants had peptide fragment(s) that were rarely found in the host proteins and (ii) the relationship between low similarity and epitope definition appears potentially applicable to T-cell determinants. The data confirmed the hypothesis that low-sequence similarity shapes or determines the epitope definition at the molecular level and provides a potential tool for designing new approaches to prevent, diagnose, and treat tuberculosis and other infectious diseases. PMID:20625421

  12. Disinfecting endoscopes: how not to transmit Mycobacterium tuberculosis by bronchoscopy.

    PubMed Central

    Leers, W D

    1980-01-01

    Mycobacterium tuberculosis was cultured from the bronchial washings of two patients who underwent bronchoscopy consecutively with the same bronchoscope. Active pulmonary tuberculosis was later confirmed in the first patient, whereas the second patient had clinical and serologic evidence of infection with respiratory syncytial virus. The bronchoscope had been cleaned with an iodophor disinfectant, which had not destroyed the tubercle bacilli. The agent recommended for chemical disinfection of fibreoptic bronchoscopes is 2% glutaraldehyde solution; the instrument should be immersed in it for 10 to 30 minutes. Five hours' exposure to ethylene oxide is recommended for sterilization of instruments. These procedures must be preceded by adequate mechanical cleaning. Then transmission of pathogenic organisms during endoscopy, which can result in nosocomial disease, misdiagnosis or inappropriate treatment, will be avoided. Images FIG. 1 FIG. 2 FIG. 3 PMID:6790150

  13. Tuberculosis

    MedlinePlus

    Tuberculosis (TB) is a disease caused by bacteria called Mycobacterium tuberculosis. The bacteria usually attack the lungs, but they can also damage other parts of the body. TB spreads through the air when a person with ...

  14. Genetic Diversity and Dynamic Distribution of Mycobacterium tuberculosis Isolates Causing Pulmonary and Extrapulmonary Tuberculosis in Thailand

    PubMed Central

    Srilohasin, Prapaporn; Tokunaga, Katsushi; Nishida, Nao; Prammananan, Therdsak; Smittipat, Nat; Mahasirimongkol, Surakameth; Chaiyasirinroje, Boonchai; Yanai, Hideki; Palittapongarnpim, Prasit

    2014-01-01

    This study examined the genetic diversity and dynamicity of circulating Mycobacterium tuberculosis strains in Thailand using nearly neutral molecular markers. The single nucleotide polymorphism (SNP)-based genotypes of 1,414 culture-positive M. tuberculosis isolates from 1,282 pulmonary tuberculosis (PTB) and 132 extrapulmonary TB (EPTB) patients collected from 1995 to 2011 were characterized. Among the eight SNP cluster groups (SCG), SCG2 (44.1%), which included the Beijing (BJ) genotype, and SCG1 (39.4%), an East African Indian genotype, were dominant. Comparisons between the genotypes of M. tuberculosis isolates causing PTB and EPTB in HIV-negative cases revealed similar prevalence trends although genetic diversity was higher in the PTB patients. The identification of 10 reported sequence types (STs) and three novel STs was hypothesized to indicate preferential expansion of the SCG2 genotype, especially the modern BJ ST10 (15.6%) and ancestral BJ ST19 (13.1%). An association between SCG2 and SCG1 genotypes and particular patient age groups implies the existence of different genetic advantages among the bacterial populations. The results revealed that increasing numbers of young patients were infected with M. tuberculosis SCGs 2 and 5, which contrasts with the reduction of the SCG1 genotype. Our results indicate the selection and dissemination of potent M. tuberculosis genotypes in this population. The determination of heterogeneity and dynamic population changes of circulating M. tuberculosis strains in countries using the Mycobacterium bovis BCG (bacillus Calmette-Guérin) vaccine are beneficial for vaccine development and control strategies. PMID:25297330

  15. An acidic sphingomyelinase Type C activity from Mycobacterium tuberculosis.

    PubMed

    Castro-Garza, Jorge; González-Salazar, Francisco; Quinn, Frederick D; Karls, Russell K; De La Garza-Salinas, Laura Hermila; Guzmán-de la Garza, Francisco J; Vargas-Villarreal, Javier

    2016-01-01

    Sphingomyelinases (SMases) catalyze the hydrolysis of sphingomyelin to ceramide and phosphorylcholine. Sphingolipids are recognized as diverse and dynamic regulators of a multitude of cellular processes mediating cell cycle control, differentiation, stress response, cell migration, adhesion, and apoptosis. Bacterial SMases are virulence factors for several species of pathogens. Whole cell extracts of Mycobacterium tuberculosis strains H37Rv and CDC1551 were assayed using [N-methyl-(14)C]-sphingomyelin as substrate. Acidic Zn(2+)-dependent SMase activity was identified in both strains. Peak SMase activity was observed at pH 5.5. Interestingly, overall SMase activity levels from CDC1551 extracts are approximately 1/3 of those of H37Rv. The presence of exogenous SMase produced by M. tuberculosis during infection may interfere with the normal host inflammatory response thus allowing the establishment of infection and disease development. This Type C activity is different from previously identified M. tuberculosis SMases. Defining the biochemical characteristics of M. tuberculosis SMases helps to elucidate the roles that these enzymes play during infection and disease. PMID:26948102

  16. LAG3 Expression in Active Mycobacterium tuberculosis Infections

    PubMed Central

    Phillips, Bonnie L.; Mehra, Smriti; Ahsan, Muhammad H.; Selman, Moises; Khader, Shabaana A.; Kaushal, Deepak

    2016-01-01

    Mycobacterium tuberculosis (MTB) is a highly successful pathogen because of its ability to persist in human lungs for long periods of time. MTB modulates several aspects of the host immune response. Lymphocyte-activation gene 3 (LAG3) is a protein with a high affinity for the CD4 receptor and is expressed mainly by regulatory T cells with immunomodulatory functions. To understand the function of LAG3 during MTB infection, a nonhuman primate model of tuberculosis, which recapitulates key aspects of natural human infection in rhesus macaques (Macaca mulatta), was used. We show that the expression of LAG3 is highly induced in the lungs and particularly in the granulomatous lesions of macaques experimentally infected with MTB. Furthermore, we show that LAG3 expression is not induced in the lungs and lung granulomas of animals exhibiting latent tuberculosis infection. However, simian immunodeficiency virus–induced reactivation of latent tuberculosis infection results in an increased expression of LAG3 in the lungs. This response is not observed in nonhuman primates infected with non-MTB bacterial pathogens, nor with simian immunodeficiency virus alone. Our data show that LAG3 was expressed primarily on CD4+ T cells, presumably by regulatory T cells but also by natural killer cells. The expression of LAG3 coincides with high bacterial burdens and changes in the host type 1 helper T-cell response. PMID:25549835

  17. Crosstalk between Mycobacterium tuberculosis and the host cell

    PubMed Central

    Dey, Bappaditya; Bishai, William R.

    2014-01-01

    The successful establishment and maintenance of a bacterial infection depends on the pathogen’s ability to subvert the host cell’s defense response and successfully survive, proliferate, or persist within the infected cell. To circumvent host defense systems, bacterial pathogens produce a variety of virulence factors that potentiate bacterial adherence and invasion and usurp host cell signaling cascades that regulate intracellular microbial survival and trafficking. Mycobacterium tuberculosis, probably one of the most successful pathogens on earth, has coexisted with humanity for centuries, and this intimate and persistent connection between these two organisms suggests that the pathogen has evolved extensive mechanisms to evade the human immune system at multiple levels. While some of these mechanisms are mediated by factors released by M. tuberculosis, others rely on host components that are hijacked to prevent the generation of an effective immune response thus benefiting the survival of M. tuberculosis within the host cell. Here, we describe several of these mechanisms, with an emphasis on the cyclic nucleotide signaling and subversion of host responses that occur at the intracellular level when tubercle bacilli encounter macrophages, a cell that becomes a safe-house for M. tuberculosis although it is specialized to kill most microbes. PMID:25303934

  18. Native New Zealand plants with inhibitory activity towards Mycobacterium tuberculosis

    PubMed Central

    2010-01-01

    Background Plants have long been investigated as a source of antibiotics and other bioactives for the treatment of human disease. New Zealand contains a diverse and unique flora, however, few of its endemic plants have been used to treat tuberculosis. One plant, Laurelia novae-zelandiae, was reportedly used by indigenous Maori for the treatment of tubercular lesions. Methods Laurelia novae-zelandiae and 44 other native plants were tested for direct anti-bacterial activity. Plants were extracted with different solvents and extracts screened for inhibition of the surrogate species, Mycobacterium smegmatis. Active plant samples were then tested for bacteriostatic activity towards M. tuberculosis and other clinically-important species. Results Extracts of six native plants were active against M. smegmatis. Many of these were also inhibitory towards M. tuberculosis including Laurelia novae-zelandiae (Pukatea). M. excelsa (Pohutukawa) was the only plant extract tested that was active against Staphylococcus aureus. Conclusions Our data provide support for the traditional use of Pukatea in treating tuberculosis. In addition, our analyses indicate that other native plant species possess antibiotic activity. PMID:20537175

  19. Gamma Interferon Release Assays for Detection of Mycobacterium tuberculosis Infection

    PubMed Central

    Denkinger, Claudia M.; Kik, Sandra V.; Rangaka, Molebogeng X.; Zwerling, Alice; Oxlade, Olivia; Metcalfe, John Z.; Cattamanchi, Adithya; Dowdy, David W.; Dheda, Keertan; Banaei, Niaz

    2014-01-01

    SUMMARY Identification and treatment of latent tuberculosis infection (LTBI) can substantially reduce the risk of developing active disease. However, there is no diagnostic gold standard for LTBI. Two tests are available for identification of LTBI: the tuberculin skin test (TST) and the gamma interferon (IFN-γ) release assay (IGRA). Evidence suggests that both TST and IGRA are acceptable but imperfect tests. They represent indirect markers of Mycobacterium tuberculosis exposure and indicate a cellular immune response to M. tuberculosis. Neither test can accurately differentiate between LTBI and active TB, distinguish reactivation from reinfection, or resolve the various stages within the spectrum of M. tuberculosis infection. Both TST and IGRA have reduced sensitivity in immunocompromised patients and have low predictive value for progression to active TB. To maximize the positive predictive value of existing tests, LTBI screening should be reserved for those who are at sufficiently high risk of progressing to disease. Such high-risk individuals may be identifiable by using multivariable risk prediction models that incorporate test results with risk factors and using serial testing to resolve underlying phenotypes. In the longer term, basic research is necessary to identify highly predictive biomarkers. PMID:24396134

  20. Prospective Genotyping of Mycobacterium tuberculosis from Fresh Clinical Samples

    PubMed Central

    Bidovec-Stojkovič, Urška; Seme, Katja; Žolnir-Dovč, Manca; Supply, Philip

    2014-01-01

    Shorter time-to-result is key for improving molecular-guided epidemiological investigation of tuberculosis (TB) cases. We performed a prospective study to evaluate the use of standardized MIRU-VNTR (mycobacterial interspersed repetitive-unit-variable-number tandem-repeat) typing of Mycobacterium tuberculosis directly on 79 fresh clinical samples from 26 TB patients consecutively enrolled over a 17-month period. Overall, complete 24-locus types were obtained for 18 out of the 26 (69.2%) patients and 14 of the 16 grade 3+ and grade 2+ samples (87.5%). The degree of completion of the genotypes obtained significantly correlated with smear microscopy grade both for 26 first samples (p = 0.0003) and for 53 follow-up samples (p = 0.002). For 20 of the 26 patients for whom complete or even incomplete M. tuberculosis isolate genotypes were obtained, typing applied to the clinical samples allowed the same unambiguous conclusions regarding case clustering or uniqueness as those that could have been drawn based on the corresponding cultured isolates. Standard 24 locus MIRU-VNTR typing of M. tuberculosis can be applied directly to fresh clinical samples, with typeability depending on the bacterial load in the sample. PMID:25313883

  1. Thioridazine Alters the Cell-Envelope Permeability of Mycobacterium tuberculosis.

    PubMed

    de Keijzer, Jeroen; Mulder, Arnout; de Haas, Petra E W; de Ru, Arnoud H; Heerkens, Evy M; Amaral, Leonard; van Soolingen, Dick; van Veelen, Peter A

    2016-06-01

    The increasing occurrence of multidrug resistant tuberculosis exerts a major burden on treatment of this infectious disease. Thioridazine, previously used as a neuroleptic, is active against extensively drug resistant tuberculosis when added to other second- and third-line antibiotics. By quantitatively studying the proteome of thioridazine-treated Mycobacterium tuberculosis, we discovered the differential abundance of several proteins that are involved in the maintenance of the cell-envelope permeability barrier. By assessing the accumulation of fluorescent dyes in mycobacterial cells over time, we demonstrate that long-term drug exposure of M. tuberculosis indeed increased the cell-envelope permeability. The results of the current study demonstrate that thioridazine induced an increase in cell-envelope permeability and thereby the enhanced uptake of compounds. These results serve as a novel explanation to the previously reported synergistic effects between thioridazine and other antituberculosis drugs. This new insight in the working mechanism of this antituberculosis compound could open novel perspectives of future drug-administration regimens in combinational therapy. PMID:27068340

  2. Profiling the Proteome of Mycobacterium tuberculosis during Dormancy and Reactivation.

    PubMed

    Gopinath, Vipin; Raghunandanan, Sajith; Gomez, Roshna Lawrence; Jose, Leny; Surendran, Arun; Ramachandran, Ranjit; Pushparajan, Akhil Raj; Mundayoor, Sathish; Jaleel, Abdul; Kumar, Ramakrishnan Ajay

    2015-08-01

    Tuberculosis, caused by Mycobacterium tuberculosis, still remains a major global health problem. The main obstacle in eradicating this disease is the ability of this pathogen to remain dormant in macrophages, and then reactivate later under immuno-compromised conditions. The physiology of hypoxic nonreplicating M. tuberculosis is well-studied using many in vitro dormancy models. However, the physiological changes that take place during the shift from dormancy to aerobic growth (reactivation) have rarely been subjected to a detailed investigation. In this study, we developed an in vitro reactivation system by re-aerating the virulent laboratory strain of M. tuberculosis that was made dormant employing Wayne's dormancy model, and compared the proteome profiles of dormant and reactivated bacteria using label-free one-dimensional LC/MS/MS analysis. The proteome of dormant bacteria was analyzed at nonreplicating persistent stage 1 (NRP1) and stage 2 (NRP2), whereas that of reactivated bacteria was analyzed at 6 and 24 h post re-aeration. Proteome of normoxially grown bacteria served as the reference. In total, 1871 proteins comprising 47% of the M. tuberculosis proteome were identified, and many of them were observed to be expressed differentially or uniquely during dormancy and reactivation. The number of proteins detected at different stages of dormancy (764 at NRP1, 691 at NRP2) and reactivation (768 at R6 and 983 at R24) was very low compared with that of the control (1663). The number of unique proteins identified during normoxia, NRP1, NRP2, R6, and R24 were 597, 66, 56, 73, and 94, respectively. We analyzed various biological functions during these conditions. Fluctuation in the relative quantities of proteins involved in energy metabolism during dormancy and reactivation was the most significant observation we made in this study. Proteins that are up-regulated or uniquely expressed during reactivation from dormancy offer to be attractive targets for therapeutic

  3. Essential Metabolites of Mycobacterium tuberculosis and Their Mimics

    PubMed Central

    Lamichhane, Gyanu; Freundlich, Joel S.; Ekins, Sean; Wickramaratne, Niluka; Nolan, Scott T.; Bishai, William R.

    2011-01-01

    An organism requires a range of biomolecules for its growth. By definition, these are essential molecules which constitute the basic metabolic requirements of an organism. A small organic molecule with chemical similarity to that of an essential metabolite may bind to the enzyme that catalyzes its production and inhibit it, likely resulting in the stasis or death of the organism. Here, we report a high-throughput approach for identifying essential metabolites of an organism using genetic and biochemical approaches and then implement computational approaches to identify metabolite mimics. We generated and genotyped 5,126 Mycobacterium tuberculosis mutants and performed a statistical analysis to determine putative essential genes. The essential molecules of M. tuberculosis were classified as products of enzymes that are encoded by genes in this list. Although incomplete, as many enzymes of M. tuberculosis have yet to be identified and characterized, this is the first report of a large number of essential molecules of the organism. We identified essential metabolites of three distinct metabolic pathways in M. tuberculosis and selected molecules with chemical similarity using cheminformatics strategies that illustrate a variety of different pharmacophores. Our approach is aimed at systematic identification of essential molecules and their mimics as a blueprint for development of effective chemical probes of M. tuberculosis metabolism, with the ultimate goal of seeking drugs that can kill this pathogen. As an illustration of this approach, we report that compounds JFD01307SC and l-methionine-S-sulfoximine, which share chemical similarity with an essential molecule of M. tuberculosis, inhibited the growth of this organism at micromolar concentrations. PMID:21285434

  4. Energy Metabolism and Drug Efflux in Mycobacterium tuberculosis

    PubMed Central

    Black, Philippa A.; Warren, Robin M.; Louw, Gail E.; van Helden, Paul D.; Victor, Thomas C.

    2014-01-01

    The inherent drug susceptibility of microorganisms is determined by multiple factors, including growth state, the rate of drug diffusion into and out of the cell, and the intrinsic vulnerability of drug targets with regard to the corresponding antimicrobial agent. Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), remains a significant source of global morbidity and mortality, further exacerbated by its ability to readily evolve drug resistance. It is well accepted that drug resistance in M. tuberculosis is driven by the acquisition of chromosomal mutations in genes encoding drug targets/promoter regions; however, a comprehensive description of the molecular mechanisms that fuel drug resistance in the clinical setting is currently lacking. In this context, there is a growing body of evidence suggesting that active extrusion of drugs from the cell is critical for drug tolerance. M. tuberculosis encodes representatives of a diverse range of multidrug transporters, many of which are dependent on the proton motive force (PMF) or the availability of ATP. This suggests that energy metabolism and ATP production through the PMF, which is established by the electron transport chain (ETC), are critical in determining the drug susceptibility of M. tuberculosis. In this review, we detail advances in the study of the mycobacterial ETC and highlight drugs that target various components of the ETC. We provide an overview of some of the efflux pumps present in M. tuberculosis and their association, if any, with drug transport and concomitant effects on drug resistance. The implications of inhibiting drug extrusion, through the use of efflux pump inhibitors, are also discussed. PMID:24614376

  5. Population genomics of Mycobacterium tuberculosis in the Inuit.

    PubMed

    Lee, Robyn S; Radomski, Nicolas; Proulx, Jean-Francois; Levade, Ines; Shapiro, B Jesse; McIntosh, Fiona; Soualhine, Hafid; Menzies, Dick; Behr, Marcel A

    2015-11-01

    Nunavik, Québec suffers from epidemic tuberculosis (TB), with an incidence 50-fold higher than the Canadian average. Molecular studies in this region have documented limited bacterial genetic diversity among Mycobacterium tuberculosis isolates, consistent with a founder strain and/or ongoing spread. We have used whole-genome sequencing on 163 M. tuberculosis isolates from 11 geographically isolated villages to provide a high-resolution portrait of bacterial genetic diversity in this setting. All isolates were lineage 4 (Euro-American), with two sublineages present (major, n = 153; minor, n = 10). Among major sublineage isolates, there was a median of 46 pairwise single-nucleotide polymorphisms (SNPs), and the most recent common ancestor (MRCA) was in the early 20th century. Pairs of isolates within a village had significantly fewer SNPs than pairs from different villages (median: 6 vs. 47, P < 0.00005), indicating that most transmission occurs within villages. There was an excess of nonsynonymous SNPs after the diversification of M. tuberculosis within Nunavik: The ratio of nonsynonymous to synonymous substitution rates (dN/dS) was 0.534 before the MRCA but 0.777 subsequently (P = 0.010). Nonsynonymous SNPs were detected across all gene categories, arguing against positive selection and toward genetic drift with relaxation of purifying selection. Supporting the latter possibility, 28 genes were partially or completely deleted since the MRCA, including genes previously reported to be essential for M. tuberculosis growth. Our findings indicate that the epidemiologic success of M. tuberculosis in this region is more likely due to an environment conducive to TB transmission than a particularly well-adapted strain. PMID:26483462

  6. Profiling the Proteome of Mycobacterium tuberculosis during Dormancy and Reactivation*

    PubMed Central

    Gopinath, Vipin; Raghunandanan, Sajith; Gomez, Roshna Lawrence; Jose, Leny; Surendran, Arun; Ramachandran, Ranjit; Pushparajan, Akhil Raj; Mundayoor, Sathish; Jaleel, Abdul; Kumar, Ramakrishnan Ajay

    2015-01-01

    Tuberculosis, caused by Mycobacterium tuberculosis, still remains a major global health problem. The main obstacle in eradicating this disease is the ability of this pathogen to remain dormant in macrophages, and then reactivate later under immuno-compromised conditions. The physiology of hypoxic nonreplicating M. tuberculosis is well-studied using many in vitro dormancy models. However, the physiological changes that take place during the shift from dormancy to aerobic growth (reactivation) have rarely been subjected to a detailed investigation. In this study, we developed an in vitro reactivation system by re-aerating the virulent laboratory strain of M. tuberculosis that was made dormant employing Wayne's dormancy model, and compared the proteome profiles of dormant and reactivated bacteria using label-free one-dimensional LC/MS/MS analysis. The proteome of dormant bacteria was analyzed at nonreplicating persistent stage 1 (NRP1) and stage 2 (NRP2), whereas that of reactivated bacteria was analyzed at 6 and 24 h post re-aeration. Proteome of normoxially grown bacteria served as the reference. In total, 1871 proteins comprising 47% of the M. tuberculosis proteome were identified, and many of them were observed to be expressed differentially or uniquely during dormancy and reactivation. The number of proteins detected at different stages of dormancy (764 at NRP1, 691 at NRP2) and reactivation (768 at R6 and 983 at R24) was very low compared with that of the control (1663). The number of unique proteins identified during normoxia, NRP1, NRP2, R6, and R24 were 597, 66, 56, 73, and 94, respectively. We analyzed various biological functions during these conditions. Fluctuation in the relative quantities of proteins involved in energy metabolism during dormancy and reactivation was the most significant observation we made in this study. Proteins that are up-regulated or uniquely expressed during reactivation from dormancy offer to be attractive targets for therapeutic

  7. Identification of outer membrane proteins of Mycobacterium tuberculosis.

    PubMed

    Song, Houhui; Sandie, Reatha; Wang, Ying; Andrade-Navarro, Miguel A; Niederweis, Michael

    2008-11-01

    The cell wall of mycobacteria includes an unusual outer membrane of extremely low permeability. While Escherichia coli uses more than 60 proteins to functionalize its outer membrane, only two mycobacterial outer membrane proteins (OMPs) are known. The porin MspA of Mycobacterium smegmatis provided the proof of principle that integral mycobacterial OMPs share the beta-barrel structure, the absence of hydrophobic alpha-helices and the presence of a signal peptide with OMPs of gram-negative bacteria. These properties were exploited in a multi-step bioinformatic approach to predict OMPs of M. tuberculosis. A secondary structure analysis was performed for 587 proteins of M. tuberculosis predicted to be exported. Scores were calculated for the beta-strand content and the amphiphilicity of the beta-strands. Reference OMPs of gram-negative bacteria defined threshold values for these parameters that were met by 144 proteins of unknown function of M. tuberculosis. Two of them were verified as OMPs by a novel two-step experimental approach. Rv1698 and Rv1973 were detected only in the total membrane fraction of M. bovis BCG in Western blot experiments, while proteinase K digestion of whole cells showed the surface accessibility of these proteins. These findings established that Rv1698 and Rv1973 are indeed localized in the outer membrane and tripled the number of known OMPs of M. tuberculosis. Significantly, these results provide evidence for the usefulness of the bioinformatic approach to predict mycobacterial OMPs and indicate that M. tuberculosis likely has many OMPs with beta-barrel structure. Our findings pave the way to identify the set of proteins which functionalize the outer membrane of M. tuberculosis. PMID:18439872

  8. Population genomics of Mycobacterium tuberculosis in the Inuit

    PubMed Central

    Lee, Robyn S.; Radomski, Nicolas; Proulx, Jean-Francois; Levade, Ines; Shapiro, B. Jesse; McIntosh, Fiona; Soualhine, Hafid; Menzies, Dick; Behr, Marcel A.

    2015-01-01

    Nunavik, Québec suffers from epidemic tuberculosis (TB), with an incidence 50-fold higher than the Canadian average. Molecular studies in this region have documented limited bacterial genetic diversity among Mycobacterium tuberculosis isolates, consistent with a founder strain and/or ongoing spread. We have used whole-genome sequencing on 163 M. tuberculosis isolates from 11 geographically isolated villages to provide a high-resolution portrait of bacterial genetic diversity in this setting. All isolates were lineage 4 (Euro-American), with two sublineages present (major, n = 153; minor, n = 10). Among major sublineage isolates, there was a median of 46 pairwise single-nucleotide polymorphisms (SNPs), and the most recent common ancestor (MRCA) was in the early 20th century. Pairs of isolates within a village had significantly fewer SNPs than pairs from different villages (median: 6 vs. 47, P < 0.00005), indicating that most transmission occurs within villages. There was an excess of nonsynonymous SNPs after the diversification of M. tuberculosis within Nunavik: The ratio of nonsynonymous to synonymous substitution rates (dN/dS) was 0.534 before the MRCA but 0.777 subsequently (P = 0.010). Nonsynonymous SNPs were detected across all gene categories, arguing against positive selection and toward genetic drift with relaxation of purifying selection. Supporting the latter possibility, 28 genes were partially or completely deleted since the MRCA, including genes previously reported to be essential for M. tuberculosis growth. Our findings indicate that the epidemiologic success of M. tuberculosis in this region is more likely due to an environment conducive to TB transmission than a particularly well-adapted strain. PMID:26483462

  9. N,C-capped dipeptides with selectivity for mycobacterial proteasome over human proteasomes: Role of S3 and S1 binding pockets

    PubMed Central

    Chidawanyika, Tamutenda; Tsu, Christopher; Warrier, Thulasi; Vaubourgeix, Julien; Blackburn, Christopher; Gigstad, Kenneth; Sintchak, Michael; Dick, Lawrence

    2013-01-01

    We identified N,C-capped dipeptides that are selective for the Mycobacterium tuberculosis proteasome over human constitutive and immunoproteasomes. Differences in S3 and S1 binding pockets appeared to account for species-selectivity. The inhibitors are able to penetrate mycobacteria and kill non-replicating M. tuberculosis under nitrosative stress. PMID:23782398

  10. Mean Platelet Volume in Mycobacterium tuberculosis Infection

    PubMed Central

    Lee, Min Young; Kim, Young Jin; Lee, Hee Joo; Park, Tae Sung

    2016-01-01

    Introduction. Mean platelet volume (MPV) has been thought as a useful index of platelet activation. It is supposed that MPV is also associated with several inflammatory and infectious diseases. Korea still has a high incidence of tuberculosis (TB). The aim of this study was to investigate MPV as an inflammatory marker in TB patients. Materials and Methods. MPV were determined in 221 patients with TB and 143 individuals for control group. MPV was estimated by an Advia 2120 (Siemens Healthcare Diagnostics, Tarrytown, NY, USA). Results. In the TB patient group, a positive correlation was found between CRP and MPV. Age and MPV had a positive correlation in TB patient group. Conclusions. We conclude that there is a significant relation between MPV and inflammatory conditions. MPV can be an inflammatory marker to determine the disease activity in TB patients. PMID:27419136

  11. Prime–Boost with Mycobacterium smegmatis Recombinant Vaccine Improves Protection in Mice Infected with Mycobacterium tuberculosis

    PubMed Central

    Junqueira-Kipnis, Ana Paula; de Oliveira, Fábio Muniz; Trentini, Monalisa Martins; Tiwari, Sangeeta; Chen, Bing; Resende, Danilo Pires; Silva, Bruna D. S.; Chen, Mei; Tesfa, Lydia; Jacobs, William R.; Kipnis, André

    2013-01-01

    The development of a new vaccine as a substitute for Bacillus Calmette–Guerin or to improve its efficacy is one of the many World Health Organization goals to control tuberculosis. Mycobacterial vectors have been used successfully in the development of vaccines against tuberculosis. To enhance the potential utility of Mycobacterium smegmatis as a vaccine, it was transformed with a recombinant plasmid containing the partial sequences of the genes Ag85c, MPT51, and HspX (CMX) from M. tuberculosis. The newly generated recombinant strain mc2-CMX was tested in a murine model of infection. The recombinant vaccine induced specific IgG1 or IgG2a responses to CMX. CD4+ and CD8+ T cells from the lungs and spleen responded ex vivo to CMX, producing IFN-γ, IL17, TNF-α, and IL2. The vaccine thus induced a significant immune response in mice. Mice vaccinated with mc2-CMX and challenged with M. tuberculosis showed better protection than mice immunized with wild-type M. smegmatis or BCG. To increase the safety and immunogenicity of the CMX antigens, we used a recombinant strain of M. smegmatis, IKE (immune killing evasion), to express CMX. The recombinant vaccine IKE-CMX induced a better protective response than mc2-CMX. The data presented here suggest that the expression of CMX antigens improves the immune response and the protection induced in mice when M. smegmatis is used as vaccine against tuberculosis. PMID:24250805

  12. Accurate Detection of Rifampicin-Resistant Mycobacterium Tuberculosis Strains.

    PubMed

    Song, Keum-Soo; Nimse, Satish Balasaheb; Kim, Hee Jin; Yang, Jeongseong; Kim, Taisun

    2016-01-01

    In 2013 alone, the death rate among the 9.0 million people infected with Mycobacterium tuberculosis (TB) worldwide was around 14%, which is unacceptably high. An empiric treatment of patients infected with TB or drug-resistant Mycobacterium tuberculosis (MDR-TB) strain can also result in the spread of MDR-TB. The diagnostic tools which are rapid, reliable, and have simple experimental protocols can significantly help in decreasing the prevalence rate of MDR-TB strain. We report the evaluation of the 9G technology based 9G DNAChips that allow accurate detection and discrimination of TB and MDR-TB-RIF. One hundred and thirteen known cultured samples were used to evaluate the ability of 9G DNAChip in the detection and discrimination of TB and MDR-TB-RIF strains. Hybridization of immobilized probes with the PCR products of TB and MDR-TB-RIF strains allow their detection and discrimination. The accuracy of 9G DNAChip was determined by comparing its results with sequencing analysis and drug susceptibility testing. Sequencing analysis showed 100% agreement with the results of 9G DNAChip. The 9G DNAChip showed very high sensitivity (95.4%) and specificity (100%). PMID:26999135

  13. Accurate Detection of Rifampicin-Resistant Mycobacterium Tuberculosis Strains

    PubMed Central

    Song, Keum-Soo; Nimse, Satish Balasaheb; Kim, Hee Jin; Yang, Jeongseong; Kim, Taisun

    2016-01-01

    In 2013 alone, the death rate among the 9.0 million people infected with Mycobacterium tuberculosis (TB) worldwide was around 14%, which is unacceptably high. An empiric treatment of patients infected with TB or drug-resistant Mycobacterium tuberculosis (MDR-TB) strain can also result in the spread of MDR-TB. The diagnostic tools which are rapid, reliable, and have simple experimental protocols can significantly help in decreasing the prevalence rate of MDR-TB strain. We report the evaluation of the 9G technology based 9G DNAChips that allow accurate detection and discrimination of TB and MDR-TB-RIF. One hundred and thirteen known cultured samples were used to evaluate the ability of 9G DNAChip in the detection and discrimination of TB and MDR-TB-RIF strains. Hybridization of immobilized probes with the PCR products of TB and MDR-TB-RIF strains allow their detection and discrimination. The accuracy of 9G DNAChip was determined by comparing its results with sequencing analysis and drug susceptibility testing. Sequencing analysis showed 100% agreement with the results of 9G DNAChip. The 9G DNAChip showed very high sensitivity (95.4%) and specificity (100%). PMID:26999135

  14. A High-Throughput Cidality Screen for Mycobacterium Tuberculosis

    PubMed Central

    Kaur, Parvinder; Ghosh, Anirban; Krishnamurthy, Ramya Vadageri; Bhattacharjee, Deepa Gagwani; Achar, Vijayashree; Datta, Santanu; Narayanan, Shridhar; Anbarasu, Anand; Ramaiah, Sudha

    2015-01-01

    Exposure to Mycobacterium tuberculosis (Mtb) aerosols is a major threat to tuberculosis (TB) researchers, even in bio-safety level-3 (BSL-3) facilities. Automation and high-throughput screens (HTS) in BSL3 facilities are essential for minimizing manual aerosol-generating interventions and facilitating TB research. In the present study, we report the development and validation of a high-throughput, 24-well ‘spot-assay’ for selecting bactericidal compounds against Mtb. The bactericidal screen concept was first validated in the fast-growing surrogate Mycobacterium smegmatis (Msm) and subsequently confirmed in Mtb using the following reference anti-tubercular drugs: rifampicin, isoniazid, ofloxacin and ethambutol (RIOE, acting on different targets). The potential use of the spot-assay to select bactericidal compounds from a large library was confirmed by screening on Mtb, with parallel plating by the conventional gold standard method (correlation, r2 = 0.808). An automated spot-assay further enabled an MBC90 determination on resistant and sensitive Mtb clinical isolates. The implementation of the spot-assay in kinetic screens to enumerate residual Mtb after either genetic silencing (anti-sense RNA, AS-RNA) or chemical inhibition corroborated its ability to detect cidality. This relatively simple, economical and quantitative HTS considerably minimized the bio-hazard risk and enabled the selection of novel vulnerable Mtb targets and mycobactericidal compounds. Thus, spot-assays have great potential to impact the TB drug discovery process. PMID:25693161

  15. Acquired Drug Resistance in Mycobacterium tuberculosis and Poor Outcomes among Patients with Multidrug-Resistant Tuberculosis

    PubMed Central

    Kipiani, Maia; Mirtskhulava, Veriko; Tukvadze, Nestani; Magee, Matthew J.; Blumberg, Henry M.

    2015-01-01

    Rates and risk factors for acquired drug resistance and association with outcomes among patients with multidrug-resistant tuberculosis (MDR TB) are not well defined. In an MDR TB cohort from the country of Georgia, drug susceptibility testing for second-line drugs (SLDs) was performed at baseline and every third month. Acquired resistance was defined as any SLD whose status changed from susceptible at baseline to resistant at follow-up. Among 141 patients, acquired resistance in Mycobacterium tuberculosis was observed in 19 (14%); prevalence was 9.1% for ofloxacin and 9.8% for capreomycin or kanamycin. Baseline cavitary disease and resistance to >6 drugs were associated with acquired resistance. Patients with M. tuberculosis that had acquired resistance were at significantly increased risk for poor treatment outcome compared with patients without these isolates (89% vs. 36%; p<0.01). Acquired resistance occurs commonly among patients with MDR TB and impedes successful treatment outcomes. PMID:25993036

  16. Mycobacterium tuberculosis Infection in a Green-Winged Macaw (Ara chloroptera): Report with Public Health Implications

    PubMed Central

    Washko, Rita M.; Hoefer, Heidi; Kiehn, Timothy E.; Armstrong, Donald; Dorsinville, Guy; Frieden, Thomas R.

    1998-01-01

    Mycobacterium tuberculosis was isolated from the eyelid, skin, tongue, and lungs of a green-winged macaw (Ara chloroptera). Two persons living in the same household were culture positive for pulmonary tuberculosis 3 to 4 years before tuberculosis was diagnosed in the bird. Although humans have not been shown to acquire tuberculosis from birds, an infected bird may be a sentinel for human infection. PMID:9542945

  17. Mycobacterium tuberculosis and Copper: A Newly Appreciated Defense against an Old Foe?*

    PubMed Central

    Darwin, K. Heran

    2015-01-01

    Several independent studies have recently converged upon the conclusion that the human bacterial pathogen Mycobacterium tuberculosis encounters copper during infections. At least three independently regulated pathways respond to excess copper and are required for the full virulence of M. tuberculosis in animals. In this review, I will discuss the functions of the best-characterized copper-responsive proteins in M. tuberculosis, the potential sources of copper during an infection, and remaining questions about the interface between copper and tuberculosis. PMID:26055711

  18. Analysis of the Mycobacterium tuberculosis 85A antigen promoter region.

    PubMed Central

    Kremer, L; Baulard, A; Estaquier, J; Content, J; Capron, A; Locht, C

    1995-01-01

    A mycobacterial expression-secretion vector was constructed in which the Escherichia coli alkaline phosphatase (phoA) reporter gene was placed under the control of the Mycobacterium tuberculosis 85A promoter and secretion signal sequences. In recombinant Mycobacterium smegmatis and Mycobacterium bovis BCG, PhoA activity could readily be detected on the mycobacterial cell surface and in the culture supernatant, indicating that the 85A signals can drive heterologous expression and secretion in both species. In contrast to the mycobacteria, the 85A promoter did not function in E. coli. We mapped the promoter region by progressive deletions using BAL 31 exonuclease and by primer extension analysis. Insertion and deletion mutations within the promoter region indicated that, unlike most E. coli promoters but similar to Streptomyces promoters, the position of the putative -35 region was not critical for efficient promoter activity. In addition, we investigated the ability of the identified signals to drive the production and secretion in BCG of recombinant Schistosoma mansoni glutathione S-transferase (Sm28GST), a protective antigen against schistosomiasis. BALB/c mice immunized with the recombinant BCG by a single dose exhibited a weak but specific T-cell response to Sm28GST. PMID:7836298

  19. Novel Multiplex Real-Time PCR Diagnostic Assay for Identification and Differentiation of Mycobacterium tuberculosis, Mycobacterium canettii, and Mycobacterium tuberculosis Complex Strains▿†

    PubMed Central

    Reddington, Kate; O'Grady, Justin; Dorai-Raj, Siobhan; Maher, Majella; van Soolingen, Dick; Barry, Thomas

    2011-01-01

    Tuberculosis (TB) in humans is caused by members of the Mycobacterium tuberculosis complex (MTC). Rapid detection of the MTC is necessary for the timely initiation of antibiotic treatment, while differentiation between members of the complex may be important to guide the appropriate antibiotic treatment and provide epidemiological information. In this study, a multiplex real-time PCR diagnostics assay using novel molecular targets was designed to identify the MTC while simultaneously differentiating between M. tuberculosis and M. canettii. The lepA gene was targeted for the detection of members of the MTC, the wbbl1 gene was used for the differentiation of M. tuberculosis and M. canettii from the remainder of the complex, and a unique region of the M. canettii genome, a possible novel region of difference (RD), was targeted for the specific identification of M. canettii. The multiplex real-time PCR assay was tested using 125 bacterial strains (64 MTC isolates, 44 nontuberculosis mycobacteria [NTM], and 17 other bacteria). The assay was determined to be 100% specific for the mycobacteria tested. Limits of detection of 2.2, 2.17, and 0.73 cell equivalents were determined for M. tuberculosis/M. canettii, the MTC, and M. canettii, respectively, using probit regression analysis. Further validation of this diagnostics assay, using clinical samples, should demonstrate its potential for the rapid, accurate, and sensitive diagnosis of TB caused by M. tuberculosis, M. canettii, and the other members of the MTC. PMID:21123525

  20. Molecular Epidemiology of Mycobacterium tuberculosis Isolates in 100 Patients With Tuberculosis Using Pulsed Field Gel Electrophoresis

    PubMed Central

    Pooideh, Mohammad; Jabbarzadeh, Ismail; Ranjbar, Reza; Saifi, Mahnaz

    2015-01-01

    Background: Tuberculosis (TB) is a widespread infectious disease. Today, TB has created a public health crisis in the world. Genotyping of Mycobacterium tuberculosis isolates is useful for surveying the dynamics of TB infection, identifying new outbreaks, and preventing the disease. Different molecular methods for clustering of M. tuberculosis isolates have been used. Objectives: During a one year study of genotyping, 100 M. tuberculosis isolates from patients referred to Pasteur Institute of Iran were collected and their genotyping was accomplished using pulsed field gel electrophoresis (PFGE) method. Materials and Methods: Identification of all M. tuberculosis isolates was accomplished using standard biochemical and species-specific polymerase chain reaction (PCR) methods. Antibiotic susceptibility tests were performed using proportional method. After preparing PFGE plaques for each isolate of M. tuberculosis, XbaI restriction enzyme was applied for genome digestion. Finally, the digested DNA fragments were separated on 1% agarose gel and analyzed with GelCompar II software. Results: Genotyping of the studied isolates in comparison with the molecular weight marker revealed two common types; pulsotype A with 71 isolates and one multidrug resistant mycobacterium (MDR) case, and pulsotype B including 29 isolates and three MDR cases. No correlation between the antibiotypes and pulsotypes was observed. Conclusions: Molecular epidemiology studies of infectious diseases have been useful when bacterial isolates have been clustered in a period of time and in different geographical regions with variable antibiotic resistance patterns. In spite of high geographical differences and different antibiotic resistant patterns, low genetic diversity among the studied TB isolates may refer to the low rate of mutations in XbaI restriction sites in the mycobacterial genome. We also identified three MDR isolates in low-incidence pulsotype B, which could be disseminated and is highly

  1. Revisiting the essentiality of glutamate racemase in Mycobacterium tuberculosis.

    PubMed

    Morayya, Sapna; Awasthy, Disha; Yadav, Reena; Ambady, Anisha; Sharma, Umender

    2015-01-25

    Glutamate racemase (MurI) converts l-glutamate into d-glutamate which is an essential component of peptidoglycan in bacteria. The gene encoding glutamate racemase, murI has been shown to be essential for the growth of a number of bacterial species including Escherichia coli. However, in some Gram-positive species d-amino acid transaminase (Dat) can also convert l-glutamate into d-glutamate thus rendering MurI non-essential for growth. In a recent study the murI gene of Mycobacterium tuberculosis was shown to be non-essential. As d-glutamate is an essential component of peptidoglycan of M. tuberculosis, either Dat or MurI has to be essential for its survival. Since, a Dat encoding gene has not been reported in M. tuberculosis genome sequence, the reported non-essentiality of murI was unexplainable. In order to resolve this dilemma we tried to knockout murI in the presence of single and two copies of murI, in wild type and merodiploid strains respectively. It was found that murI could not be inactivated in the wild type background indicating that it could be an essential gene. Also, inactivation of murI could not be achieved in the presence of externally supplied d-glutamate in 7H9 medium suggesting that M. tuberculosis is unable to take up d-glutamate under the conditions tested. However we could generate murI knockout strains at high frequency when two copies of the gene were present indicating that at least one murI gene is required for cellular viability. The essential nature of MurI in M. tuberculosis H37Rv suggests that it could be a potential drug target. PMID:25447907

  2. Activity of 5-chloro-pyrazinamide in mice infected with Mycobacterium tuberculosis or Mycobacterium bovis

    PubMed Central

    Ahmad, Zahoor; Tyagi, Sandeep; Minkowski, Austin; Almeida, Deepak; Nuermberger, Eric L.; Peck, Kaitlin M.; Welch, John T.; Baughn, Anthony D.; Jacobs, Williams R.; Grosset, Jacques H.

    2012-01-01

    Background & objectives: Pyrazinamide is an essential component of first line anti-tuberculosis regimen as well as most of the second line regimens. This drug has a unique sterilizing activity against Mycobacterium tuberculosis. Its unique role in tuberculosis treatment has lead to the search and development of its structural analogues. One such analogue is 5-chloro-pyrazinamide (5-Cl-PZA) that has been tested under in vitro conditions against M. tuberculosis. The present study was designed with an aim to assess the activity of 5-Cl-PZA, alone and in combination with first-line drugs, against murine tuberculosis. Methods: The minimum inhibitory concentration (MIC) of 5-Cl-PZA in Middlebrook 7H9 broth (neutral pH) and the inhibitory titre of serum from mice that received a 300 mg/kg oral dose of 5-Cl-PZA 30 min before cardiac puncture were determined. To test the tolerability of orally administered 5-Cl-PZA, uninfected mice received doses up to 300 mg/kg for 2 wk. Four weeks after low-dose aerosol infection either with M. tuberculosis or M. bovis, mice were treated 5 days/wk with 5-Cl-PZA, at doses ranging from 37.5 to 150 mg/kg, either alone or in combination with isoniazid and rifampicin. Antimicrobial activity was assessed by colony-forming unit counts in lungs after 4 and 8 wk of treatment. Results: The MIC of 5-Cl-PZA against M. tuberculosis was between 12.5 and 25 μg/ml and the serum inhibitory titre was 1:4. Under the same experimental conditions, the MIC of pyrazinamide was >100 μg/ml and mouse serum had no inhibitory activity after a 300 mg/kg dose; 5-Cl-PZA was well tolerated in uninfected and infected mice up to 300 and 150 mg/kg, respectively. While PZA alone and in combination exhibited its usual antimicrobial activity in mice infected with M. tuberculosis and no activity in mice infected with M. bovis, 5-Cl-PZA exhibited antimicrobial activity neither in mice infected with M. tuberculosis nor in mice infected with M. bovis. Interpretation

  3. Personal respiratory protection against Mycobacterium tuberculosis.

    PubMed

    Fennelly, K P

    1997-03-01

    Although there are no data demonstrating the effectiveness of personal respiratory protection in the prevention of occupational tuberculosis, there are sound theoretical bases supporting the use of respirators to reduce the risk of inhalational exposure. The major factor that limits the effectiveness of most respirators is the leakage between the face and the mask. There are data suggesting that traditional fit testing of respirators does not adequately predict the degree of protection in actual use, and more research is needed in that area. There is a large range of infectiousness of aerosols of TB, and classes of respirators vary greatly in the degree of protection they offer. I have argued that respirator selection should be based on anticipated exposures. High-risk exposures to TB are often associated with cough-inducing procedures or with aerosolization of infected tissues during autopsies. In my opinion, the most reasonable type of respirator for such high-risk situations in health care settings is a PAPR hood. The concentration of infectious aerosols in well-ventilated respiratory isolation rooms is likely to be very low, and the new N95 respirators offer a reasonable balance of comfort, cost, practicality, and protection. Preliminary data from mathematical modeling studies suggest there may be little additional benefit from more sophisticated personal respiratory protection in such settings. Additional research is needed to more accurately assess exposures to TB, to determine the size and aerodynamic behavior of TB generated by infectious patients, and to more accurately define the role and effectiveness of personal respiratory protection against TB. PMID:9098607

  4. Immunogenicity and cross-reactivity against Mycobacterium tuberculosis of proteoliposomes derived from Mycobacterium bovis BCG.

    PubMed

    Reyes, Fátima; Tirado, Yanely; Puig, Alina; Borrero, Reinier; Reyes, Giselle; Fernández, Sonsire; Pérez, José Luis; Kadir, Ramlah; Zayas, Caridad; Norazmi, Mohd Nor; Sarmiento, María E; Acosta, Armando

    2013-01-01

    The only currently available vaccine against tuberculosis (TB) is Mycobacterium bovis Bacille Calmette-Guerin (BCG), which has inconsistent efficacy to protect against the disease in adults. M. tuberculosis (MTB) cell wall components have been implicated in the pathogenicity of TB and therefore have been a prime target for the identification and characterization of cell wall proteins with potential application in vaccine development. In this regard, proteoliposomes (PLs) derived from mycobacteria containing lipids and cell wall proteins could be potential vaccine candidates against TB. In the present study PLs derived from BCG were prepared. These homogeneous population of spherical microparticles was then immunized into Balb/c mice. Sera of immunized animals showed high IgG response and strong cross-reactivity against different MTB antigens.These results showed that BCG PLs could be potential vaccine candidates against TB. PMID:23458692

  5. Rapid susceptibility testing of Mycobacterium avium complex and Mycobacterium tuberculosis isolated from AIDS patients

    NASA Technical Reports Server (NTRS)

    Dhople, Arvind M.

    1994-01-01

    In ominous projections issued by both U.S. Public Health Service and the World Health Organization, the epidemic of HIV infection will continue to rise more rapidly worldwide than predicted earlier. The AIDS patients are susceptible to diseases called opportunistic infections of which tuberculosis and Mycobacterium avium complex (MAC) infection are most common. This has created an urgent need to uncover new drugs for the treatment of these infections. In the seventies, NASA scientists at Goddard Space Flight Center, Greenbelt, MD, had adopted a biochemical indicator, adenosine triphosphate (ATP), to detect presence of life in extraterrestrial space. We proposed to develop ATP assay technique to determine sensitivity of antibacterial compounds against MAC and M. tuberculosis.

  6. A New Approach for Pyrazinamide Susceptibility Testing in Mycobacterium tuberculosis

    PubMed Central

    Loli, Sebastian; Gilman, Robert H.; Gutierrez, Andrés; Fuentes, Patricia; Cotrina, Milagros; Kirwan, Daniela; Sheen, Patricia

    2012-01-01

    Background: Pyrazinamide (PZA) is an important drug in the treatment of tuberculosis. Microbiological methods of PZA susceptibility testing are controversial and have low reproducibility. After conversion of PZA into pyrazinoic acid (POA) by the bacterial pyrazinamidase enzyme, the drug is expelled from the bacteria by an efflux pump. Objective: To evaluate the rate of POA extrusion from Mycobacterium tuberculosis as a parameter to detect PZA resistance. Methods: The rate of POA extrusion and PZA susceptibility determined by BACTEC 460 were measured for 34 strains in a previous study. PZA resistance was modeled in a logistic regression with the pyrazinoic efflux rate. Result: POA efflux rate predicted PZA resistance with 70.83%–92.85% sensitivity and 100% specificity compared with BACTEC 460. Conclusion: POA efflux rate could be a useful tool for predicting PZA resistance in M. tuberculosis. Further exploration of this approach may lead to the development of new tools for diagnosing PZA resistance, which may be of public health importance. PMID:22372927

  7. Direct inhibitors of InhA active against Mycobacterium tuberculosis

    PubMed Central

    Manjunatha, Ujjini H.; Rao, Srinivasa P. S.; Kondreddi, Ravinder Reddy; Noble, Christian G.; Camacho, Luis R.; Tan, Bee H.; Ng, Seow H.; Ng, Pearly Shuyi; Ma, N. L.; Lakshminarayana, Suresh B.; Herve, Maxime; Barnes, S. Whitney; Yu, Weixuan; Kuhen, Kelli; Blasco, Francesca; Beer, David; Walker, John R.; Tonge, Peter J.; Glynne, Richard; Smith, Paul W.; Diagana, Thierry T.

    2015-01-01

    New chemotherapeutic agents are urgently required to combat the global spread of multi-drug resistant tuberculosis (MDR-TB). The mycobacterial enoyl reductase, InhA, is one of the few clinically-validated targets in tuberculosis drug discovery. Here, we report the identification of a new class of direct InhA inhibitors, the 4-hydroxy-2-pyridones, using phenotypic high-throughput whole-cell screening. This class of orally-active compounds showed potent bactericidal activity against common isoniazid-resistant TB clinical isolates. Biophysical studies revealed that 4-hydroxy-2-pyridones bound specifically to InhA in an NADH-dependent manner and blocked the enoyl-substrate binding pocket. The lead compound NITD-916 directly blocked InhA in a dose-dependent manner and showed in vivo efficacy in acute and established mouse models of infection by Mycobacterium tuberculosis. Collectively, our structural and biochemical data open up new avenues for rational structure-guided optimization of the 4-hydroxy-2-pyridone class of compounds for the treatment of MDR-TB. PMID:25568071

  8. EFFECT OF PYRAZINAMIDASE ACTIVITY ON PYRAZINAMIDE RESISTANCE IN MYCOBACTERIUM TUBERCULOSIS

    PubMed Central

    Sheen, Patricia; Ferrer, Patricia; Gilman, Robert H.; López-Llano, Jon; Fuentes, Patricia; Valencia, Eddy; Zimic, Mirko J.

    2009-01-01

    Resistance of Mycobacterium tuberculosis to pyrazinamide is associated with mutations in the pncA gene, which codes for pyrazinamidase. The association between the enzymatic activity of mutated pyrazinamidases and the level of pyrazinamide resistance remains poorly understood. Twelve M. tuberculosis clinical isolates resistant to pyrazinamide were selected based on Wayne activity and localization of pyrazinamidase mutation. Recombinant pyrazinamidases were expressed and tested for their kinetic parameters (activity, kcat, Km, and efficiency). Pyrazinamide resistance level was measured by Bactec-460TB and 7H9 culture. The linear correlation between the resistance level and the kinetic parameters of the corresponding mutated pyrazinamidase was calculated. The enzymatic activity and efficiency of the mutated pyrazinamidases varied with the site of mutation and ranged widely from low to high levels close to the corresponding of the wild-type enzyme. The level of resistance was significantly associated with pyrazinamidase activity and efficiency, but only 27.3% of its statistical variability was explained. Although pyrazinamidase mutations are indeed associated with resistance, the loss of pyrazinamidase activity and efficiency as assessed in the recombinant mutated enzymes is not sufficient to explain a high variability of the level of pyrazinamide resistance, suggesting that complementary mechanisms for pyrazinamide resistance in M. tuberculosis with mutations in pncA are more important than currently thought. PMID:19249243

  9. Genotyping of ancient Mycobacterium tuberculosis strains reveals historic genetic diversity

    PubMed Central

    Müller, Romy; Roberts, Charlotte A.; Brown, Terence A.

    2014-01-01

    The evolutionary history of the Mycobacterium tuberculosis complex (MTBC) has previously been studied by analysis of sequence diversity in extant strains, but not addressed by direct examination of strain genotypes in archaeological remains. Here, we use ancient DNA sequencing to type 11 single nucleotide polymorphisms and two large sequence polymorphisms in the MTBC strains present in 10 archaeological samples from skeletons from Britain and Europe dating to the second–nineteenth centuries AD. The results enable us to assign the strains to groupings and lineages recognized in the extant MTBC. We show that at least during the eighteenth–nineteenth centuries AD, strains of M. tuberculosis belonging to different genetic groups were present in Britain at the same time, possibly even at a single location, and we present evidence for a mixed infection in at least one individual. Our study shows that ancient DNA typing applied to multiple samples can provide sufficiently detailed information to contribute to both archaeological and evolutionary knowledge of the history of tuberculosis. PMID:24573854

  10. Proteomic analysis of ofloxacin-mono resistant Mycobacterium tuberculosis isolates.

    PubMed

    Lata, Manju; Sharma, Divakar; Deo, Nirmala; Tiwari, Pramod Kumar; Bisht, Deepa; Venkatesan, Krishnamurthy

    2015-09-01

    Drug resistance particularly, multi drug resistance tuberculosis (MDR-TB) has emerged as a major problem in the chemotherapy of tuberculosis. Ofloxacin (OFX) has been used as second-line drug against MDR-TB. The principal target of the OFX is DNA gyrase encoded by gyrA and gyrB genes. Many explanations have been proposed for drug resistance to OFX but still some mechanisms are unknown. As proteins manifest most of the biological processes, these are attractive targets for developing drugs and diagnostics/therapeutics. We examined the OFX resistant Mycobacterium tuberculosis isolates by proteomic approach (2DE-MALDI-TOF-MS) and bioinformatic tools under OFX induced conditions. Our study showed fourteen proteins (Rv0685, Rv0363c, Rv2744c, Rv3803c, Rv2534c, Rv2140c, Rv1475c, Rv0440, Rv2245, Rv1436, Rv3551, Rv0148, Rv2882c and Rv0733) with increased intensities in OFX resistant and OFX induced as compared to susceptible isolates. Bioinformatic analysis of hypothetical proteins (Rv2744c, Rv2140c, Rv3551 and Rv0148) revealed the presence of conserved motifs and domains. Molecular docking showed proper interaction of OFX with residues of conserved motifs. These proteins might be involved in the OFX modulation/neutralization and act as novel resistance mechanisms as well as potential for diagnostics and drug targets against OFX resistance. This article is part of a Special Issue entitled: Proteomics in India. PMID:26238929

  11. Copper resistance is essential for virulence of Mycobacterium tuberculosis.

    PubMed

    Wolschendorf, Frank; Ackart, David; Shrestha, Tej B; Hascall-Dove, Laurel; Nolan, Scott; Lamichhane, Gyanu; Wang, Ying; Bossmann, Stefan H; Basaraba, Randall J; Niederweis, Michael

    2011-01-25

    Copper (Cu) is essential for many biological processes, but is toxic when present in excessive amounts. In this study, we provide evidence that Cu plays a crucial role in controlling tuberculosis. A Mycobacterium tuberculosis (Mtb) mutant lacking the outer membrane channel protein Rv1698 accumulated 100-fold more Cu and was more susceptible to Cu toxicity than WT Mtb. Similar phenotypes were observed for a M. smegmatis mutant lacking the homolog Ms3747, demonstrating that these mycobacterial copper transport proteins B (MctB) are essential for Cu resistance and maintenance of low intracellular Cu levels. Guinea pigs responded to infection with Mtb by increasing the Cu concentration in lung lesions. Loss of MctB resulted in a 1,000- and 100-fold reduced bacterial burden in lungs and lymph nodes, respectively, in guinea pigs infected with Mtb. In mice, the persistence defect of the Mtb mctB mutant was exacerbated by the addition of Cu to the diet. These experiments provide evidence that Cu is used by the mammalian host to control Mtb infection and that Cu resistance mechanisms are crucial for Mtb virulence. Importantly, Mtb is much more susceptible to Cu than other bacteria and is killed in vitro by Cu concentrations lower than those found in phagosomes of macrophages. Hence, this study reveals an Achilles heel of Mtb that might be a promising target for tuberculosis chemotherapy. PMID:21205886

  12. Pulmonary Tuberculosis Caused by Mycobacterium bovis in China

    PubMed Central

    Jiang, Guanglu; Wang, Guirong; Chen, Suting; Yu, Xia; Wang, Xiaobo; Zhao, Liping; Ma, Yifeng; Dong, Lingling; Huang, Hairong

    2015-01-01

    The epidemiology of Mycobacterium bovis infection in humans in China is unknown. In this study, pulmonary tuberculosis caused by M. bovis in China was studied. A total of 4069 clinical strains isolated from sputa during the 2007–2009 nationwide surveillance of drug-resistant tuberculosis in China were analyzed. M. bovis was identified by para-nitrobenzoic acid and thiophen-2-carboxylic acid hydrazide growth tests, spoligotyping and multiplex PCR amplification. In addition, a total of 1828 clinical specimens were recruited from Beijing Chest Hospital (Beijing, China) for Löwenstein-Jensen (LJ) culture, both on standard LJ medium and LJ medium containing 4.5 mg/ml(W/V) sodium pyruvate, the latter being the preferred medium for M. bovis growth. The isolates which demonstrated more vigorous on pyruvate containing medium than on standard LJ medium were then identified by multiplex PCR amplification. Only 1 isolate from the nationwide surveillance was confirmed as M. bovis-BCG. The isolate belonged to a predominant spoligotype SB0120 (ST482). In addition, no M. bovis isolate was acquired by the continuous screening step in Beijing Chest Hospital. M. bovis has a negligible contribution to pulmonary tuberculosis in China, so neither laboratory identification nor clinical treatment of M. bovis infection need be considered in routine work. PMID:25736338

  13. Isolation and restriction site maps of the genes encoding five Mycobacterium tuberculosis proteins.

    PubMed

    Shinnick, T M; Krat, C; Schadow, S

    1987-07-01

    A series of recombinant phage expressing five Mycobacterium tuberculosis antigens were isolated. Restriction maps for these antigens were deduced, and the size of the expressed proteins was determined. PMID:3036711

  14. Isolation and restriction site maps of the genes encoding five Mycobacterium tuberculosis proteins.

    PubMed Central

    Shinnick, T M; Krat, C; Schadow, S

    1987-01-01

    A series of recombinant phage expressing five Mycobacterium tuberculosis antigens were isolated. Restriction maps for these antigens were deduced, and the size of the expressed proteins was determined. Images PMID:3036711

  15. Racial differences in susceptibility to infection by Mycobacterium tuberculosis.

    PubMed

    Stead, W W; Senner, J W; Reddick, W T; Lofgren, J P

    1990-02-15

    The prevalence of tuberculosis among blacks is known to be about twice that among whites. When we looked at infection rates among the initially tuberculin-negative residents of 165 racially integrated nursing homes in Arkansas, we were stimulated to investigate whether this difference could be due in part to racial differences in susceptibility to Mycobacterium tuberculosis infection. A new infection was defined by an increase of greater than or equal to 12 mm of induration after a tuberculin skin test (5 tuberculin units) administered at least 60 days after a negative two-step test. On repeat skin testing of the 25,398 initially tuberculin-negative nursing home residents, we found that 13.8 percent of the blacks and only 7.2 percent of the whites had evidence of a new infection (relative risk, 1.9; 95 percent confidence interval, 1.7 to 2.1). Blacks were infected more frequently, regardless of the race of the source patient. In homes with a single source patient who was white, 17.4 percent of the black and 11.7 percent of the white residents became infected (relative risk, 1.5; 95 percent confidence interval, 1.2 to 1.9); in homes with a single source patient who was black, 12.4 percent of the black and 7.7 percent of the white residents became infected (relative risk, 1.6; 95 percent confidence interval, 1.2 to 2.1). However, there was no racial difference in the percentage of residents who had recently converted to positive status who, in the absence of preventive therapy, were later found to have clinical tuberculosis (blacks, 11.5 percent; whites, 10.6 percent). Data from three outbreaks of tuberculosis in two prisons also showed that blacks have about twice the relative risk of whites of becoming infected with M. tuberculosis. We conclude that blacks are more readily infected by M. tuberculosis than are whites. The data also suggest that susceptibility to M. tuberculosis infection varies independently of the factors governing the progression to clinical

  16. Multiple small RNAs identified in Mycobacterium bovis BCG are also expressed in Mycobacterium tuberculosis and Mycobacterium smegmatis

    PubMed Central

    DiChiara, Jeanne M.; Contreras-Martinez, Lydia M.; Livny, Jonathan; Smith, Dorie; McDonough, Kathleen A.; Belfort, Marlene

    2010-01-01

    Tuberculosis (TB) is a major global health problem, infecting millions of people each year. The causative agent of TB, Mycobacterium tuberculosis, is one of the world’s most ancient and successful pathogens. However, until recently, no work on small regulatory RNAs had been performed in this organism. Regulatory RNAs are found in all three domains of life, and have already been shown to regulate virulence in well-known pathogens, such as Staphylococcus aureus and Vibrio cholera. Here we report the discovery of 34 novel small RNAs (sRNAs) in the TB-complex M. bovis BCG, using a combination of experimental and computational approaches. Putative homologues of many of these sRNAs were also identified in M. tuberculosis and/or M. smegmatis. Those sRNAs that are also expressed in the non-pathogenic M. smegmatis could be functioning to regulate conserved cellular functions. In contrast, those sRNAs identified specifically in M. tuberculosis could be functioning in mediation of virulence, thus rendering them potential targets for novel antimycobacterials. Various features and regulatory aspects of some of these sRNAs are discussed. PMID:20181675

  17. Mycobacterium tuberculosis PPE protein Rv0256c induces strong B cell response in tuberculosis patients.

    PubMed

    Abraham, Philip Raj; Latha, Gaddam Suman; Valluri, Vijaya Lakshmi; Mukhopadhyay, Sangita

    2014-03-01

    Tuberculosis (TB) is one of the most important diseases of humans and major public health problem worldwide. Early and accurate diagnosis of TB is necessary for the treatment, prevention, and control of TB. Therefore, it is important to identify suitable antigens that can differentiate active tuberculosis patients from BCG-vaccinated individuals. In the present study, we have used Rv0256c (PPE2) protein of Mycobacterium tuberculosis to screen the sera of infected patients belonging to different clinical TB presentations, and BCG-vaccinated clinically healthy individuals by enzyme immunoassay. Our results demonstrated that Rv0256c displayed stronger and specific immunoreactivity against the sera obtained from clinically active tuberculosis patients compared to PPD and ESAT-6 and could differentiate the TB-patients from the BCG-vaccinated controls. Importantly, Rv0256c was also found to detect even the extrapulmonary and smear-negative pulmonary cases which often are tedious and difficult to detect using conventional diagnostic methods. This study suggests that Rv0256c can be used as a potential marker for the serodiagnosis of tuberculosis patients. PMID:23827809

  18. Systems-Based Approaches to Probing Metabolic Variation within the Mycobacterium tuberculosis Complex

    PubMed Central

    Lofthouse, Emma K.; Wheeler, Paul R.; Beste, Dany J. V.; Khatri, Bhagwati L.; Wu, Huihai; Mendum, Tom A.; Kierzek, Andrzej M.; McFadden, Johnjoe

    2013-01-01

    The Mycobacterium tuberculosis complex includes bovine and human strains of the tuberculosis bacillus, including Mycobacterium tuberculosis, Mycobacterium bovis and the Mycobacterium bovis BCG vaccine strain. M. bovis has evolved from a M. tuberculosis-like ancestor and is the ancestor of the BCG vaccine. The pathogens demonstrate distinct differences in virulence, host range and metabolism, but the role of metabolic differences in pathogenicity is poorly understood. Systems biology approaches have been used to investigate the metabolism of M. tuberculosis, but not to probe differences between tuberculosis strains. In this study genome scale metabolic networks of M. bovis and M. bovis BCG were constructed and interrogated, along with a M. tuberculosis network, to predict substrate utilisation, gene essentiality and growth rates. The models correctly predicted 87-88% of high-throughput phenotype data, 75-76% of gene essentiality data and in silico-predicted growth rates matched measured rates. However, analysis of the metabolic networks identified discrepancies between in silico predictions and in vitro data, highlighting areas of incomplete metabolic knowledge. Additional experimental studies carried out to probe these inconsistencies revealed novel insights into the metabolism of these strains. For instance, that the reduction in metabolic capability observed in bovine tuberculosis strains, as compared to M. tuberculosis, is not reflected by current genetic or enzymatic knowledge. Hence, the in silico networks not only successfully simulate many aspects of the growth and physiology of these mycobacteria, but also provide an invaluable tool for future metabolic studies. PMID:24098743

  19. Comparative analyses of transport proteins encoded within the genomes of Mycobacterium tuberculosis and Mycobacterium leprae

    PubMed Central

    Youm, Jiwon; Saier, Milton H.

    2012-01-01

    The co-emergence of multidrug resistant pathogenic bacterial strains and the HIV pandemic has made tuberculosis a leading public health threat. The causative agent is Mycobacterium tuberculosis (Mtu), a facultative intracellular parasite. Mycobacterium leprae (Mle), a related organism that causes leprosy, is an obligate intracellular parasite. Given that different transporters are required for bacterial growth and persistence under a variety of growth conditions, we conducted comparative analyses of transport proteins encoded within the genomes of these two organisms. A minimal set of genes required for intracellular and extracellular life were identified. Drug efflux systems utilizing primary active transport mechanisms have been preferentially retained in Mle and still others preferentially lost. Transporters associated with environmental adaptation found in Mtu were mostly lost in Mle. These findings provide starting points for experimental studies that may elucidate the dependencies of pathogenesis on transport for these two pathogenic mycobacteria. They also lead to suggestions regarding transporters that function in intra- versus extra-cellular growth. PMID:22179038

  20. Rv2358 and FurB: Two Transcriptional Regulators from Mycobacterium tuberculosis Which Respond to Zinc

    PubMed Central

    Canneva, Fabio; Branzoni, Manuela; Riccardi, Giovanna; Provvedi, Roberta; Milano, Anna

    2005-01-01

    In a previous work, we demonstrated that the Mycobacterium tuberculosis Rv2358-furB operon is induced by zinc. In this study, the orthologous genes from Mycobacterium smegmatis mc2155 were inactivated and mutants analyzed. Rv2358 protein was purified and found to bind upstream of the Rv2358 gene. Binding was inhibited by Zn2+ ions. PMID:16077132

  1. Potent Inhibitors of a Shikimate Pathway Enzyme from Mycobacterium tuberculosis

    PubMed Central

    Reichau, Sebastian; Jiao, Wanting; Walker, Scott R.; Hutton, Richard D.; Baker, Edward N.; Parker, Emily J.

    2011-01-01

    Tuberculosis remains a serious global health threat, with the emergence of multidrug-resistant strains highlighting the urgent need for novel antituberculosis drugs. The enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyzes the first step of the shikimate pathway for the biosynthesis of aromatic compounds. This pathway has been shown to be essential in Mycobacterium tuberculosis, the pathogen responsible for tuberculosis. DAH7PS catalyzes a condensation reaction between P-enolpyruvate and erythrose 4-phosphate to give 3-deoxy-d-arabino-heptulosonate 7-phosphate. The enzyme reaction mechanism is proposed to include a tetrahedral intermediate, which is formed by attack of an active site water on the central carbon of P-enolpyruvate during the course of the reaction. Molecular modeling of this intermediate into the active site reported in this study shows a configurational preference consistent with water attack from the re face of P-enolpyruvate. Based on this model, we designed and synthesized an inhibitor of DAH7PS that mimics this reaction intermediate. Both enantiomers of this intermediate mimic were potent inhibitors of M. tuberculosis DAH7PS, with inhibitory constants in the nanomolar range. The crystal structure of the DAH7PS-inhibitor complex was solved to 2.35 Å. Both the position of the inhibitor and the conformational changes of active site residues observed in this structure correspond closely to the predictions from the intermediate modeling. This structure also identifies a water molecule that is located in the appropriate position to attack the re face of P-enolpyruvate during the course of the reaction, allowing the catalytic mechanism for this enzyme to be clearly defined. PMID:21454647

  2. Molecular diagnosis of fluoroquinolone resistance in Mycobacterium tuberculosis.

    PubMed

    Bernard, Christine; Veziris, Nicolas; Brossier, Florence; Sougakoff, Wladimir; Jarlier, Vincent; Robert, Jérôme; Aubry, Alexandra

    2015-03-01

    As a consequence of the use of fluoroquinolones (FQ), resistance to FQ has emerged, leading to cases of nearly untreatable and extensively drug-resistant tuberculosis. Mutations in DNA gyrase represent the main mechanism of FQ resistance. A full understanding of the pattern of mutations found in FQ-resistant (FQ(r)) clinical isolates, and of their proportions, is crucial for improving molecular methods for the detection of FQ resistance in Mycobacterium tuberculosis. In this study, we reviewed the detection of FQ resistance in isolates addressed to the French National Reference Center for Mycobacteria from 2007 to 2012, with the aim of evaluating the performance of PCR sequencing in a real-life context. gyrA and gyrB sequencing, performed prospectively on M. tuberculosis clinical isolates, was compared for FQ susceptibility to 2 mg/liter ofloxacin by the reference proportion method. A total of 605 isolates, of which 50% were multidrug resistant, were analyzed. The increase in FQ(r) strains among multidrug-resistant (MDR) strains during the time of the study was alarming (8% to 30%). The majority (78%) of the isolates with gyrA mutations were FQ(r), whereas only 36% of those with gyrB mutations were FQ(r). Only 12% of the FQ(r) isolates had a single mutation in gyrB. Combined gyrA and gyrB sequencing led to >93% sensitivity for detecting resistance. The analysis of the four false-positive and the five false-negative results of gyrA and gyrB sequencing illustrated the actual limitations of the reference proportion method. Our data emphasize the need for combined gyrA and gyrB sequencing in the investigation of FQ susceptibility in M. tuberculosis and challenge the validity of the current phenotype-based approach as the diagnostic gold standard for determining FQ resistance. PMID:25534742

  3. Immunogenic potential of latency associated antigens against Mycobacterium tuberculosis.

    PubMed

    Singh, Swati; Saraav, Iti; Sharma, Sadhna

    2014-02-01

    Tuberculosis remains a great health threat to the world among infectious diseases particularly with the advent of human immunodeficiency virus and emergence of drug resistant strains. In the light of the inconsistent efficacy imparted by the only currently available pre-exposure vaccine bacillus Calmette-Guerin BCG, the development of an improved TB vaccine is a very high international research priority. Vaccine candidates currently in clinical trials are also pre-exposure vaccines that aim to prevent active tuberculosis during an individual's lifetime. According to World Health Organization approximately a third of the world's population is latently infected with Mycobacterium tuberculosis. Dormancy or latency of Mycobacteria is associated with the formation of granuloma with poorly perfused interior leading to expression of genes which help them survive in this hostile environment. A group of about 50 genes belonging to the DosR regulon also known as latency antigens are expressed by Mycobacteria when they are persisting in the immuno-competent host. An understanding of the immunological effects produced by products of these latency induced genes may help in making a more potent vaccine. Incorporation of latency antigens into improved (live or subunit) vaccines may enhance the impact of these vaccines in which BCG priming can be followed by multisubunit protein boosting. These vaccines could act as post exposure vaccines for containment and prevention of latent TB activation. This heterologous boosting of BCG-primed immunity will be able to stimulate the known immune correlates of protective immunity against M. tuberculosis i.e. TH1 cells (CD4(+) and CD8(+) T cells) mediated immune responses with cytokines such as IFN-γ and TNF-α⋅ In our review we have analysed and compared the immunogenic potential of various latency-associated antigens of the DosR regulon in line with the current strategy of developing a recombinant post exposure booster vaccine. PMID

  4. Rapid Diagnosis of Tuberculosis by Real-Time High-Resolution Imaging of Mycobacterium tuberculosis Colonies

    PubMed Central

    Ghodbane, Ramzi; Asmar, Shady; Betzner, Marlena; Linet, Marie; Pierquin, Joseph; Raoult, Didier

    2015-01-01

    Culture remains the cornerstone of diagnosis for pulmonary tuberculosis, but the fastidiousness of Mycobacterium tuberculosis may delay culture-based diagnosis for weeks. We evaluated the performance of real-time high-resolution imaging for the rapid detection of M. tuberculosis colonies growing on a solid medium. A total of 50 clinical specimens, including 42 sputum specimens, 4 stool specimens, 2 bronchoalveolar lavage fluid specimens, and 2 bronchial aspirate fluid specimens were prospectively inoculated into (i) a commercially available Middlebrook broth and evaluated for mycobacterial growth indirectly detected by measuring oxygen consumption (standard protocol) and (ii) a home-made solid medium incubated in an incubator featuring real-time high-resolution imaging of colonies (real-time protocol). Isolates were identified by Ziehl-Neelsen staining and matrix-assisted laser desorption ionization–time of flight mass spectrometry. Use of the standard protocol yielded 14/50 (28%) M. tuberculosis isolates, which is not significantly different from the 13/50 (26%) M. tuberculosis isolates found using the real-time protocol (P = 1.00 by Fisher's exact test), and the contamination rate of 1/50 (2%) was not significantly different from the contamination rate of 2/50 (4%) using the real-time protocol (P = 1.00). The real-time imaging protocol showed a 4.4-fold reduction in time to detection, 82 ± 54 h versus 360 ± 142 h (P < 0.05). These preliminary data give the proof of concept that real-time high-resolution imaging of M. tuberculosis colonies is a new technology that shortens the time to growth detection and the laboratory diagnosis of pulmonary tuberculosis. PMID:26085608

  5. Rapid Diagnosis of Tuberculosis by Real-Time High-Resolution Imaging of Mycobacterium tuberculosis Colonies.

    PubMed

    Ghodbane, Ramzi; Asmar, Shady; Betzner, Marlena; Linet, Marie; Pierquin, Joseph; Raoult, Didier; Drancourt, Michel

    2015-08-01

    Culture remains the cornerstone of diagnosis for pulmonary tuberculosis, but the fastidiousness of Mycobacterium tuberculosis may delay culture-based diagnosis for weeks. We evaluated the performance of real-time high-resolution imaging for the rapid detection of M. tuberculosis colonies growing on a solid medium. A total of 50 clinical specimens, including 42 sputum specimens, 4 stool specimens, 2 bronchoalveolar lavage fluid specimens, and 2 bronchial aspirate fluid specimens were prospectively inoculated into (i) a commercially available Middlebrook broth and evaluated for mycobacterial growth indirectly detected by measuring oxygen consumption (standard protocol) and (ii) a home-made solid medium incubated in an incubator featuring real-time high-resolution imaging of colonies (real-time protocol). Isolates were identified by Ziehl-Neelsen staining and matrix-assisted laser desorption ionization-time of flight mass spectrometry. Use of the standard protocol yielded 14/50 (28%) M. tuberculosis isolates, which is not significantly different from the 13/50 (26%) M. tuberculosis isolates found using the real-time protocol (P = 1.00 by Fisher's exact test), and the contamination rate of 1/50 (2%) was not significantly different from the contamination rate of 2/50 (4%) using the real-time protocol (P = 1.00). The real-time imaging protocol showed a 4.4-fold reduction in time to detection, 82 ± 54 h versus 360 ± 142 h (P < 0.05). These preliminary data give the proof of concept that real-time high-resolution imaging of M. tuberculosis colonies is a new technology that shortens the time to growth detection and the laboratory diagnosis of pulmonary tuberculosis. PMID:26085608

  6. Inactivation of Mycobacterium paratuberculosis and Mycobacterium tuberculosis in fresh soft cheese by gamma radiation

    NASA Astrophysics Data System (ADS)

    Badr, Hesham M.

    2011-11-01

    The effectiveness of gamma irradiation on the inactivation of Mycobacterium paratuberculosis, Mycobacterium bovis and Mycobacterium tuberculosis in fresh soft cheese that prepared from artificially inoculated milk samples was studied. Irradiation at dose of 2 kGy was sufficient for the complete inactivation of these mycobacteria as they were not detected in the treated samples during storage at 4±1 °C for 15 days. Moreover, irradiation of cheese samples, that were prepared from un-inoculated milk, at this effective dose had no significant effects on their gross composition and contents from riboflavin, niacin and pantothenic acid, while significant decreases in vitamin A and thiamin were observed. In addition, irradiation of cheese samples had no significant effects on their pH and nitrogen fractions contents, except for the contents of ammonia, which showed a slight, but significant, increases due to irradiation. The analysis of cheese fats indicated that irradiation treatment induced significant increase in their oxidation parameters and contents from free fatty acids; however, the observed increases were relatively low. On the other hand, irradiation of cheese samples induced no significant alterations on their sensory properties. Thus, irradiation dose of 2 kGy can be effectively applied to ensure the safety of soft cheese with regards to these harmful mycobacteria.

  7. Cell-autonomous effector mechanisms against mycobacterium tuberculosis.

    PubMed

    MacMicking, John D

    2014-10-01

    Few pathogens run the gauntlet of sterilizing immunity like Mycobacterium tuberculosis (Mtb). This organism infects mononuclear phagocytes and is also ingested by neutrophils, both of which possess an arsenal of cell-intrinsic effector mechanisms capable of eliminating it. Here Mtb encounters acid, oxidants, nitrosylating agents, and redox congeners, often exuberantly delivered under low oxygen tension. Further pressure is applied by withholding divalent Fe²⁺, Mn²⁺, Cu²⁺, and Zn²⁺, as well as by metabolic privation in the form of carbon needed for anaplerosis and aromatic amino acids for growth. Finally, host E3 ligases ubiquinate, cationic peptides disrupt, and lysosomal enzymes digest Mtb as part of the autophagic response to this particular pathogen. It is a testament to the evolutionary fitness of Mtb that sterilization is rarely complete, although sufficient to ensure most people infected with this airborne bacterium remain disease-free. PMID:25081628

  8. The DNA-binding network of Mycobacterium tuberculosis

    PubMed Central

    Minch, Kyle J.; Rustad, Tige R.; Peterson, Eliza J. R.; Winkler, Jessica; Reiss, David J.; Ma, Shuyi; Hickey, Mark; Brabant, William; Morrison, Bob; Turkarslan, Serdar; Mawhinney, Chris; Galagan, James E.; Price, Nathan D.; Baliga, Nitin S.; Sherman, David R.

    2015-01-01

    Mycobacterium tuberculosis (MTB) infects 30% of all humans and kills someone every 20–30 s. Here we report genome-wide binding for ~80% of all predicted MTB transcription factors (TFs), and assayed global expression following induction of each TF. The MTB DNA-binding network consists of ~16,000 binding events from 154 TFs. We identify >50 TF-DNA consensus motifs and >1,150 promoter-binding events directly associated with proximal gene regulation. An additional ~4,200 binding events are in promoter windows and represent strong candidates for direct transcriptional regulation under appropriate environmental conditions. However, we also identify >10,000 ‘dormant’ DNA-binding events that cannot be linked directly with proximal transcriptional control, suggesting that widespread DNA binding may be a common feature that should be considered when developing global models of coordinated gene expression. PMID:25581030

  9. Turning the respiratory flexibility of Mycobacterium tuberculosis against itself

    PubMed Central

    Lamprecht, Dirk A.; Finin, Peter M.; Rahman, Md. Aejazur; Cumming, Bridgette M.; Russell, Shannon L.; Jonnala, Surendranadha R.; Adamson, John H.; Steyn, Adrie J. C.

    2016-01-01

    The Mycobacterium tuberculosis (Mtb) electron transport chain (ETC) has received significant attention as a drug target, however its vulnerability may be affected by its flexibility in response to disruption. Here we determine the effect of the ETC inhibitors bedaquiline, Q203 and clofazimine on the Mtb ETC, and the value of the ETC as a drug target, by measuring Mtb's respiration using extracellular flux technology. We find that Mtb's ETC rapidly reroutes around inhibition by these drugs and increases total respiration to maintain ATP levels. Rerouting is possible because Mtb rapidly switches between terminal oxidases, and, unlike eukaryotes, is not susceptible to back pressure. Increased ETC activity potentiates clofazimine's production of reactive oxygen species, causing rapid killing in vitro and in a macrophage model. Our results indicate that combination therapy targeting the ETC can be exploited to enhance killing of Mtb. PMID:27506290

  10. A robust SNP barcode for typing Mycobacterium tuberculosis complex strains

    PubMed Central

    Coll, Francesc; McNerney, Ruth; Guerra-Assunção, José Afonso; Glynn, Judith R.; Perdigão, João; Viveiros, Miguel; Portugal, Isabel; Pain, Arnab; Martin, Nigel; Clark, Taane G.

    2014-01-01

    Strain-specific genomic diversity in the Mycobacterium tuberculosis complex (MTBC) is an important factor in pathogenesis that may affect virulence, transmissibility, host response and emergence of drug resistance. Several systems have been proposed to classify MTBC strains into distinct lineages and families. Here, we investigate single-nucleotide polymorphisms (SNPs) as robust (stable) markers of genetic variation for phylogenetic analysis. We identify ~92k SNP across a global collection of 1,601 genomes. The SNP-based phylogeny is consistent with the gold-standard regions of difference (RD) classification system. Of the ~7k strain-specific SNPs identified, 62 markers are proposed to discriminate known circulating strains. This SNP-based barcode is the first to cover all main lineages, and classifies a greater number of sublineages than current alternatives. It may be used to classify clinical isolates to evaluate tools to control the disease, including therapeutics and vaccines whose effectiveness may vary by strain type. PMID:25176035

  11. An Elucidation of Neutrophil Functions against Mycobacterium tuberculosis Infection

    PubMed Central

    Morris, Devin; Nguyen, Thien; Kim, John; Kassissa, Christine; Khurasany, Melissa; Luong, Jennifer; Kasko, Sarah; Pandya, Shalin; Chu, Michael; Chi, Po-Ting; Lagman, Minette; Venketaraman, Vishwanath

    2013-01-01

    We characterized the functions of neutrophils in response to Mycobacterium tuberculosis (M. tb) infection, with particular reference to glutathione (GSH). We examined the effects of GSH in improving the ability of neutrophils to control intracellular M. tb infection. Our findings indicate that increasing the intracellular levels of GSH with a liposomal formulation of GSH (L-GSH) resulted in reduction in the levels of free radicals and increased acidification of M. tb containing phagosomes leading to the inhibition in the growth of M. tb. This inhibitory mechanism is dependent on the presence of TNF-α and IL-6. Our studies demonstrate a novel regulatory mechanism adapted by the neutrophils to control M. tb infection. PMID:24312131

  12. Turning the respiratory flexibility of Mycobacterium tuberculosis against itself.

    PubMed

    Lamprecht, Dirk A; Finin, Peter M; Rahman, Md Aejazur; Cumming, Bridgette M; Russell, Shannon L; Jonnala, Surendranadha R; Adamson, John H; Steyn, Adrie J C

    2016-01-01

    The Mycobacterium tuberculosis (Mtb) electron transport chain (ETC) has received significant attention as a drug target, however its vulnerability may be affected by its flexibility in response to disruption. Here we determine the effect of the ETC inhibitors bedaquiline, Q203 and clofazimine on the Mtb ETC, and the value of the ETC as a drug target, by measuring Mtb's respiration using extracellular flux technology. We find that Mtb's ETC rapidly reroutes around inhibition by these drugs and increases total respiration to maintain ATP levels. Rerouting is possible because Mtb rapidly switches between terminal oxidases, and, unlike eukaryotes, is not susceptible to back pressure. Increased ETC activity potentiates clofazimine's production of reactive oxygen species, causing rapid killing in vitro and in a macrophage model. Our results indicate that combination therapy targeting the ETC can be exploited to enhance killing of Mtb. PMID:27506290

  13. Cell-Autonomous Effector Mechanisms against Mycobacterium tuberculosis

    PubMed Central

    MacMicking, John D.

    2014-01-01

    Few pathogens run the gauntlet of sterilizing immunity like Mycobacterium tuberculosis (Mtb). This organism infects mononuclear phagocytes and is also ingested by neutrophils, both of which possess an arsenal of cell-intrinsic effector mechanisms capable of eliminating it. Here Mtb encounters acid, oxidants, nitrosylating agents, and redox congeners, often exuberantly delivered under low oxygen tension. Further pressure is applied by withholding divalent Fe2+, Mn2+, Cu2+, and Zn2+, as well as by metabolic privation in the form of carbon needed for anaplerosis and aromatic amino acids for growth. Finally, host E3 ligases ubiquinate, cationic peptides disrupt, and lysosomal enzymes digest Mtb as part of the autophagic response to this particular pathogen. It is a testament to the evolutionary fitness of Mtb that sterilization is rarely complete, although sufficient to ensure most people infected with this airborne bacterium remain disease-free. PMID:25081628

  14. CCL2 Responses to Mycobacterium tuberculosis Are Associated with Disease Severity in Tuberculosis

    PubMed Central

    Hasan, Zahra; Cliff, Jacqueline M.; Dockrell, Hazel M.; Jamil, Bushra; Irfan, Muhammad; Ashraf, Mussarat; Hussain, Rabia

    2009-01-01

    Background Leucocyte activating chemokines such as CCL2, CCL3, and CXCL8 together with proinflammatory IFNγ, TNFα and downmodulatory IL10 play a central role in the restriction of M. tuberculosis infections, but is unclear whether these markers are indicative of tuberculosis disease severity. Methodology We investigated live M. tuberculosis- and M. bovis BCG- induced peripheral blood mononuclear cell responses in patients with tuberculosis (TB) and healthy endemic controls (ECs, n = 36). TB patients comprised pulmonary (PTB, n = 34) and extrapulmonary groups, subdivided into those with less severe localized extrapulmonary TB (L-ETB, n = 16) or severe disseminated ETB (D-ETB, n = 16). Secretion of CCL2, IFNγ, IL10 and CCL3, and mRNA expression of CCL2, TNFα, CCL3 and CXCL8 were determined. Results M. tuberculosis- and BCG- induced CCL2 secretion was significantly increased in both PTB and D-ETB (p<0.05, p<0.01) as compared with L-ETB patients. CCL2 secretion in response to M. tuberculosis was significantly greater than to BCG in the PTB and D-ETB groups. M. tuberculosis-induced CCL2 mRNA transcription was greater in PTB than L-ETB (p = 0.023), while CCL2 was reduced in L-ETB as compared with D-ETB (p = 0.005) patients. M. tuberculosis –induced IFNγ was greater in L-ETB than PTB (p = 0.04), while BCG-induced IFNγ was greater in L-ETB as compared with D-ETB patients (p = 0.036). TNFα mRNA expression was raised in PTB as compared with L-ETB group in response to M. tuberculosis (p = 0.02) and BCG (p = 0.03). Mycobacterium-induced CCL3 and CXCL8 was comparable between TB groups. Conclusions The increased CCL2 and TNFα in PTB patients may support effective leucocyte recruitment and M. tuberculosis localization. CCL2 alone is associated with severity of TB, possibly due to increased systemic inflammation found in severe disseminated TB or due to increased monocyte infiltration to lung parenchyma in pulmonary disease. PMID

  15. Rapid genotypic detection of rifampin- and isoniazid-resistant Mycobacterium tuberculosis directly in clinical specimens.

    PubMed

    Bang, Didi; Bengård Andersen, Ase; Thomsen, Vibeke Østergaard

    2006-07-01

    A multiplex PCR DNA strip assay (Genotype MTBDR) designed to detect rifampin (rpoB) and high-level isoniazid (katG) resistance mutations in Mycobacterium tuberculosis isolates was optimized for clinical specimens. Successful genotypic results were achieved with 36 of 38 (95%) smear-positive respiratory specimens, allowing rapid therapeutic adjustments in transmittable drug-resistant tuberculosis. PMID:16825393

  16. Draft Genome Sequence of Mycobacterium tuberculosis KT-0133, Isolated in South Korea.

    PubMed

    Kwon, Taesoo; Han, Seung Jung; Yoo, Won Gi; Yun, Mi-Ran; Lee, Sanghyun; Lee, Jong Seok; Kim, Dae-Won

    2016-01-01

    Here, we present the draft genome sequence of Mycobacterium tuberculosis KT-0133, which belongs to the Korean-Beijing family. This sequence will provide a new perspective on the evolution and accommodation of M. tuberculosis KT-0133 in human hosts. PMID:26868407

  17. Emergence of potential superbug mycobacterium tuberculosis, lessons from new delhi mutant-1 bacterial strains.

    PubMed

    Nazir, Taha; Abraham, Suraj; Islam, Azharul

    2012-01-01

    Recent reports have shown that certain bacterial strains attain the New Delhi Metallo-beta-lactamase-1 (NDM-1) enzyme and become resistant to a broad range of antibiotics. Similarly, more dangerous "superbugs" of multi-drug resistant (MDR) and extensive drug resistant (XDR) Mycobacterium tuberculosis strains are gradually emerging through rapid genetic mutation caused by prescription non-compliance or unsupervised indiscriminate use of anti-tubercular drugs or other antibiotics. Mycobacterium tuberculosis cases have been reported in highly susceptible population groups including the aboriginal communities of US and Canada. In Canada alone, the total number of reported tuberculosis cases has decreased over the past decade. However, there is a steady increase in HIV cases in certain communities including the aboriginal communities. Reintroduction of MDR/XDR strains of tuberculosis is possible in these susceptible communities, which in turn may pose serious public health situation. MDR/XDR strains of tuberculosis are virtually untreatable using current anti-tubercular medication protocols. Thus, MDR/XDR tuberculosis presents a grave global public health threat. The unpredictable genetic mechanism involved in generating MDR/XDR resistant strains of Mycobacterium tuberculosis may pose greater challenges in developing appropriate treatment strategies. In this article, we briefly review potential genetic mechanism of emerging NDM-1 bacterial strains and draw a rationale parallel to the underlying genetic mechanism of MDR/XDR Mycobacterium tuberculosis strain development. PMID:23267308

  18. Clinical Impact of Mycobacterium tuberculosis W-Beijing Genotype Strain Infection on Aged Patients in Taiwan▿

    PubMed Central

    Feng, Jia-Yih; Su, Wei-Juin; Tsai, Cheng-Chien; Chang, Shi-Chuan

    2008-01-01

    The impact of W-Beijing genotype Mycobacterium tuberculosis on treatment outcome was evaluated in 249 newly diagnosed tuberculosis patients. No significant difference in the treatment outcome was found between the W-Beijing and non-W-Beijing groups. However, a poor outcome was more common in the elderly patients (≥65 years) infected with the W-Beijing strain. PMID:18596137

  19. A cooperative oxygen-binding hemoglobin from Mycobacterium tuberculosis

    PubMed Central

    Couture, Manon; Yeh, Syun-Ru; Wittenberg, Beatrice A.; Wittenberg, Jonathan B.; Ouellet, Yannick; Rousseau, Denis L.; Guertin, Michel

    1999-01-01

    Two putative hemoglobin genes, glbN and glbO, were recently discovered in the complete genome sequence of Mycobacterium tuberculosis H37Rv. Here, we show that the glbN gene encodes a dimeric hemoglobin (HbN) that binds oxygen cooperatively with very high affinity (P50 = 0.013 mmHg at 20°C) because of a fast combination (25 μM−1⋅s−1) and a slow dissociation (0.2 s−1) rate. Resonance Raman spectroscopy and ligand association/dissociation kinetic measurements, along with mutagenesis studies, reveal that the stabilization of the bound oxygen is achieved through a tyrosine at the B10 position in the distal pocket of the heme with a conformation that is unique among the globins. Physiological studies performed with Mycobacterium bovis bacillus Calmette–Guérin demonstrate that the expression of HbN is greatly enhanced during the stationary phase in aerobic cultures but not under conditions of limited oxygen availability. The results suggest that, physiologically, the primary role of HbN may be to protect the bacilli against reactive nitrogen species produced by the host macrophage. PMID:10500158

  20. Response of inbred mice to aerosol challenge with Mycobacterium tuberculosis.

    PubMed

    Musa, S A; Kim, Y; Hashim, R; Wang, G Z; Dimmer, C; Smith, D W

    1987-08-01

    An autosomal dominant gene (Bcg), which maps to mouse chromosome 1, has been shown to confer on mice resistance to attenuated Mycobacterium bovis BCG Montreal, Salmonella typhimurium, and Leishmania donovani. Most animal models used for the study of the Bcg gene have involved intravenous injection of a large number of microorganisms (greater than 10(4) CFU). The present study examines the effect of the Bcg gene on the resistance of inbred mice to challenge via the respiratory route with 5 to 10 CFU of virulent Mycobacterium tuberculosis. The number of tubercle bacilli recovered from the lung lobes indicates that the growth kinetics of the microorganism did not differ between BCG-resistant and BCG-susceptible strains of mice. The number of tubercle bacilli recovered from the spleen was also similar among strains. Although there were reproducible differences in the time of first recovery of bacilli from the spleen, these differences appeared to be unrelated to the expression of the Bcg gene. When mice were challenged with purified protein derivative, all strains responded similarly as observed by measurements of footpad swelling. PMID:3112014

  1. [Tuberculosis cutis luposa gigantea with Mycobacterium bovis detection].

    PubMed

    Bonnekoh, B; Schütt-Gerowitt, H; Thiele, B; Mahrle, G

    1990-10-01

    In an 80-year-old woman, retired farmworker, we observed lupus vulgaris extending over more than half of her leg. The extreme size of the affected area made us talk of a giant form in this case. Bacteriological investigation revealed Mycobacterium bovis. The minimal amount of tuberculin required to induce a positive intradermal reaction was 10 IU (GT Behring). Another case with similar dimensions (reported by Christiansen in 1967) had been caused by Mycobacterium avium and developed over a period of at least 5 years. The vast cutaneous affection of our patient, in contrast, had developed within only one year, starting from a brownish macula of the size of a palm on her upper leg. This macula - presumably the manifestation of quiescent lupus vulgaris - had not changed for more than 40 years. This late exacerbation of post-primary tuberculosis might have been favored by the patient's reduced immunologic resistance on account of her advanced age. In addition, local cofactors - namely ankylosis of her knee and contact eczematous dermatitis - have to be considered. In accordance with the resistogram, the disease responded to monotherapy with isoniazide. PMID:2291294

  2. Antigenic characterization of dimorphic surface protein in Mycobacterium tuberculosis.

    PubMed

    Matsuba, Takashi; Siddiqi, Umme Ruman; Hattori, Toshio; Nakajima, Chie; Fujii, Jun; Suzuki, Yasuhiko

    2016-05-01

    The Mycobacterium tuberculosis Rv0679c protein is a surface protein that contributes to host cell invasion. We previously showed that a single nucleotide transition of the Rv0679c gene leads to a single amino acid substitution from asparagine to lysine at codon 142 in the Beijing genotype family. In this study, we examined the immunological effect of this substitution. Several recombinant proteins were expressed in Escherichia coli and Mycobacterium smegmatis and characterized with antisera and two monoclonal antibodies named 5D4-C2 and 8G10-H2. A significant reduction of antibody binding was detected by enzyme-linked immunosorbent assay (ELISA) and western blot analysis in the Lys142-type protein. This reduction of 8G10-H2 binding was more significant, with the disappearance of a signal in the proteins expressed by recombinant mycobacteria in western blot analysis. In addition, epitope mapping analysis of the recombinant proteins showed a linear epitope by 5D4-C2 and a discontinuous epitope by 8G10-H2. The antibody recognizing the conformational epitope detected only mycobacterial Asn142-type recombinant protein. Our results suggest that a single amino acid substitution of Rv0679c has potency for antigenic change in Beijing genotype strains. PMID:27190237

  3. Characterization of Mycobacterium tuberculosis EsxA Membrane Insertion

    PubMed Central

    Ma, Yue; Keil, Verena; Sun, Jianjun

    2015-01-01

    EsxA (ESAT-6), an important virulence factor of Mycobacterium tuberculosis, plays an essential role in phagosome rupture and bacterial cytosolic translocation within host macrophages. Our previous study showed that EsxA exhibits a unique membrane-interacting activity that is not found in its ortholog from nonpathogenic Mycobacterium smegmatis. However, the molecular mechanism of EsxA membrane insertion remains unknown. In this study, we generated truncated EsxA proteins with deletions of the N- and/or C-terminal flexible arm. Using a fluorescence-based liposome leakage assay, we found that both the N- and C-terminal arms were required for membrane disruption. Moreover, we found that, upon acidification, EsxA converted into a more organized structure with increased α-helical content, which was evidenced by CD analysis and intrinsic tryptophan fluorescence. Finally, using an environmentally sensitive fluorescent dye, we obtained direct evidence that the central helix-turn-helix motif of EsxA inserted into the membranes and formed a membrane-spanning pore. A model of EsxA membrane insertion is proposed and discussed. PMID:25645924

  4. STAT3 Represses Nitric Oxide Synthesis in Human Macrophages upon Mycobacterium tuberculosis Infection

    PubMed Central

    Queval, Christophe J.; Song, Ok-Ryul; Deboosère, Nathalie; Delorme, Vincent; Debrie, Anne-Sophie; Iantomasi, Raffaella; Veyron-Churlet, Romain; Jouny, Samuel; Redhage, Keely; Deloison, Gaspard; Baulard, Alain; Chamaillard, Mathias; Locht, Camille; Brodin, Priscille

    2016-01-01

    Mycobacterium tuberculosis is a successful intracellular pathogen. Numerous host innate immune responses signaling pathways are induced upon mycobacterium invasion, however their impact on M. tuberculosis replication is not fully understood. Here we reinvestigate the role of STAT3 specifically inside human macrophages shortly after M. tuberculosis uptake. We first show that STAT3 activation is mediated by IL-10 and occurs in M. tuberculosis infected cells as well as in bystander non-colonized cells. STAT3 activation results in the inhibition of IL-6, TNF-α, IFN-γ and MIP-1β. We further demonstrate that STAT3 represses iNOS expression and NO synthesis. Accordingly, the inhibition of STAT3 is detrimental for M. tuberculosis intracellular replication. Our study thus points out STAT3 as a key host factor for M. tuberculosis intracellular establishment in the early stages of macrophage infection. PMID:27384401

  5. Genetic diversity of Mycobacterium tuberculosis isolated from tuberculosis patients in the Serengeti ecosystem in Tanzania

    PubMed Central

    Mbugi, Erasto V.; Katale, Bugwesa Z.; Siame, Keith K.; Keyyu, Julius D.; Kendall, Sharon L.; Dockrell, Hazel M.; Streicher, Elizabeth M.; Michel, Anita L.; Rweyemamu, Mark M.; Warren, Robin M.; Matee, Mecky I.; van Helden, Paul D.

    2015-01-01

    Summary This study was part of a larger cross-sectional survey that was evaluating tuberculosis (TB) infection in humans, livestock and wildlife in the Serengeti ecosystem in Tanzania. The study aimed at evaluating the genetic diversity of Mycobacterium tuberculosis isolates from TB patients attending health facilities in the Serengeti ecosystem. DNA was extracted from 214 sputum cultures obtained from consecutively enrolled newly diagnosed untreated TB patients aged ≥18 years. Spacer oligonucleotide typing (spoligotyping) and Mycobacterium Interspersed Repetitive Units and Variable Number Tandem Repeat (MIRU-VNTR) were used to genotype M. tuberculosis to establish the circulating lineages. Of the214 M. tuberculosis isolates genotyped, 55 (25.7%) belonged to the Central Asian (CAS) family, 52 (24.3%) were T family (an ill-defined family), 38 (17.8%) belonged to the Latin American Mediterranean (LAM) family, 25 (11.7%) to the East-African Indian (EAI) family, 25 (11.7%) comprised of different unassigned (‘Serengeti’) strain families, while 8 (3.7%) belonged to the Beijing family. A minority group that included Haarlem, X, U and S altogether accounted for 11 (5.2%) of all genotypes. MIRU-VNTR typing produced diverse patterns within and between families indicative of unlinked transmission chains. We conclude that, in the Serengeti ecosystem only a few successful families predominate namely CAS, T, LAM and EAI families. Other types found in lower prevalence are Beijing, Haarlem, X, S and MANU. The Haarlem, EAI_Somalia, LAM3 and S/convergent and X2 subfamilies found in this study were not reported in previous studies in Tanzania. PMID:25522841

  6. The mtp40 gene is not present in all strains of Mycobacterium tuberculosis.

    PubMed Central

    Weil, A; Plikaytis, B B; Butler, W R; Woodley, C L; Shinnick, T M

    1996-01-01

    A multiple PCR-based assay that targets IS6110 and the mtp40 gene was evaluated for the rapid differentiation of Mycobacterium bovis and M. tuberculosis, two of the causative agents of tuberculosis. The IS6110 target is present in both species, whereas the mtp40 gene was thought to be specific for M.tuberculosis (P.Del Portillo, L.A. Murillo, and M.E. Patarroyo, J. Clin. Microbiol. 29:2163-2168, 1991). However, the mtp-40 gene is not present in all M. tuberculosis strains and, hence, is not useful for differentiating M.tuberculosis and M.bovis. PMID:8862608

  7. Transcriptional Adaptation of Drug-tolerant Mycobacterium tuberculosis During Treatment of Human Tuberculosis

    PubMed Central

    Walter, Nicholas D.; Dolganov, Gregory M.; Garcia, Benjamin J.; Worodria, William; Andama, Alfred; Musisi, Emmanuel; Ayakaka, Irene; Van, Tran T.; Voskuil, Martin I.; de Jong, Bouke C.; Davidson, Rebecca M.; Fingerlin, Tasha E.; Kechris, Katerina; Palmer, Claire; Nahid, Payam; Daley, Charles L.; Geraci, Mark; Huang, Laurence; Cattamanchi, Adithya; Strong, Michael; Schoolnik, Gary K.; Davis, John Lucian

    2015-01-01

    Background. Treatment initiation rapidly kills most drug-susceptible Mycobacterium tuberculosis, but a bacterial subpopulation tolerates prolonged drug exposure. We evaluated drug-tolerant bacilli in human sputum by comparing messenger RNA (mRNA) expression of drug-tolerant bacilli that survive the early bactericidal phase with treatment-naive bacilli. Methods. M. tuberculosis gene expression was quantified via reverse-transcription polymerase chain reaction in serial sputa from 17 Ugandans treated for drug-susceptible pulmonary tuberculosis. Results. Within 4 days, bacterial mRNA abundance declined >98%, indicating rapid killing. Thereafter, the rate of decline slowed >94%, indicating drug tolerance. After 14 days, 16S ribosomal RNA transcripts/genome declined 96%, indicating slow growth. Drug-tolerant bacilli displayed marked downregulation of genes associated with growth, metabolism, and lipid synthesis and upregulation in stress responses and key regulatory categories—including stress-associated sigma factors, transcription factors, and toxin-antitoxin genes. Drug efflux pumps were upregulated. The isoniazid stress signature was induced by initial drug exposure, then disappeared after 4 days. Conclusions. Transcriptional patterns suggest that drug-tolerant bacilli in sputum are in a slow-growing, metabolically and synthetically downregulated state. Absence of the isoniazid stress signature in drug-tolerant bacilli indicates that physiological state influences drug responsiveness in vivo. These results identify novel drug targets that should aid in development of novel shorter tuberculosis treatment regimens. PMID:25762787

  8. Cloning, expression and characterization of Mycobacterium tuberculosis sirR.

    PubMed

    Namwat, Wises; Somnate, Baramee; Maleehual, Dutsadee; Chareonsudjai, Sorujsiri; Lulitanond, Viraphong; Faksri, Kiatichai

    2014-05-01

    Identification of new drug targets is important for the improvement of chemotherapy for tuberculosis treatment. Metal-associated gene products are candidates for novel drug development. A Mycobacterium tuberculosis (MTB) sirR-encoded protein has been proposed, but the function of MTB SirR has not yet been elucidated. Bioinformatics analysis revealed that MTB SirR contains iron binding domains with 34%-59% similarity to previously described metal-dependent gene regulators and that the gene lies in Rv2787-sirR operon. RT-PCR revealed that the Rv2787-sirR operon is transcribed a single bicistronic mRNA. Heterologous expression, purification and characterization of recombinant MTB His-tagged SirR demonstrated a 25 kDa protein (by SDS-PAGE and immunoblotting) that exists as a dimer (native PAGE). Based on electrophoretic mobility shift assay, MTB SirR bound a cis element located at -85 bp upstream of its operon. As Rv2787-sirR operon is unique only to MTB (and M. bovis), further studies on its regulation and other functions of the encoded proteins should provide leads towards the discovery of novel anti-TB drugs. PMID:24974654

  9. SInCRe-structural interactome computational resource for Mycobacterium tuberculosis.

    PubMed

    Metri, Rahul; Hariharaputran, Sridhar; Ramakrishnan, Gayatri; Anand, Praveen; Raghavender, Upadhyayula S; Ochoa-Montaño, Bernardo; Higueruelo, Alicia P; Sowdhamini, Ramanathan; Chandra, Nagasuma R; Blundell, Tom L; Srinivasan, Narayanaswamy

    2015-01-01

    We have developed an integrated database for Mycobacterium tuberculosis H37Rv (Mtb) that collates information on protein sequences, domain assignments, functional annotation and 3D structural information along with protein-protein and protein-small molecule interactions. SInCRe (Structural Interactome Computational Resource) is developed out of CamBan (Cambridge and Bangalore) collaboration. The motivation for development of this database is to provide an integrated platform to allow easily access and interpretation of data and results obtained by all the groups in CamBan in the field of Mtb informatics. In-house algorithms and databases developed independently by various academic groups in CamBan are used to generate Mtb-specific datasets and are integrated in this database to provide a structural dimension to studies on tuberculosis. The SInCRe database readily provides information on identification of functional domains, genome-scale modelling of structures of Mtb proteins and characterization of the small-molecule binding sites within Mtb. The resource also provides structure-based function annotation, information on small-molecule binders including FDA (Food and Drug Administration)-approved drugs, protein-protein interactions (PPIs) and natural compounds that bind to pathogen proteins potentially and result in weakening or elimination of host-pathogen protein-protein interactions. Together they provide prerequisites for identification of off-target binding. PMID:26130660

  10. The mechanism of redox sensing in Mycobacterium tuberculosis.

    PubMed

    Bhat, Shabir Ahmad; Singh, Nisha; Trivedi, Abhishek; Kansal, Pallavi; Gupta, Pawan; Kumar, Ashwani

    2012-10-15

    Tuberculosis epidemics have defied constraint despite the availability of effective treatment for the past half-century. Mycobacterium tuberculosis, the causative agent of TB, is continually exposed to a number of redox stressors during its pathogenic cycle. The mechanisms used by Mtb to sense redox stress and to maintain redox homeostasis are central to the success of Mtb as a pathogen. Careful analysis of the Mtb genome has revealed that Mtb lacks classical redox sensors such as FNR, FixL, and OxyR. Recent studies, however, have established that Mtb is equipped with various sophisticated redox sensors that can detect diverse types of redox stress, including hypoxia, nitric oxide, carbon monoxide, and the intracellular redox environment. Some of these sensors, such as heme-based DosS and DosT, are unique to mycobacteria, whereas others, such as the WhiB proteins and anti-σ factor RsrA, are unique to actinobacteria. This article provides a comprehensive review of the literature on these redox-sensory modules in the context of TB pathogenesis. PMID:22921590

  11. Amperometric immunosensor for rapid detection of Mycobacterium tuberculosis

    NASA Astrophysics Data System (ADS)

    Hiraiwa, Morgan; Kim, Jong-Hoon; Lee, Hyun-Boo; Inoue, Shinnosuke; Becker, Annie L.; Weigel, Kris M.; Cangelosi, Gerard A.; Lee, Kyong-Hoon; Chung, Jae-Hyun

    2015-05-01

    Tuberculosis (TB) has been a major public health problem, which can be better controlled by using accurate and rapid diagnosis in low-resource settings. A simple, portable, and sensitive detection method is required for point-of-care (POC) settings. This paper studies an amperometric biosensor using a microtip immunoassay for a rapid and low-cost detection of Mycobacterium tuberculosis (MTB) in sputum. MTB in sputum is specifically captured on the functionalized microtip surface and detected by electric current. According to the numerical study, the current signal on the microtip surface is linearly changed with increasing immersion depth. Using a reference microtip, the immersion depth is compensated for a sensing microtip. On the microtip surface, target bacteria are concentrated and organized by a coffee-ring effect, which amplifies the electric current. To enhance the signal-to-noise ratio, both the sample processing and rinsing steps are presented with the use of deionized water as a medium for the amperometric measurement. When applied to cultured MTB cells spiked into human sputum, the detection limit was 100 CFU mL-1, comparable to a more labor-intensive fluorescence detection method reported previously.

  12. Tuberculosis Caused by Mycobacterium africanum, United States, 2004–2013

    PubMed Central

    Bloss, Emily; Heilig, Charles M.; Click, Eleanor S.

    2016-01-01

    Mycobacterium africanum is endemic to West Africa and causes tuberculosis (TB). We reviewed reported cases of TB in the United States during 2004–2013 that had lineage assigned by genotype (spoligotype and mycobacterial interspersed repetitive unit variable number tandem repeats). M. africanum caused 315 (0.4%) of 73,290 TB cases with lineage assigned by genotype. TB caused by M. africanum was associated more with persons from West Africa (adjusted odds ratio [aOR] 253.8, 95% CI 59.9–1,076.1) and US-born black persons (aOR 5.7, 95% CI 1.2–25.9) than with US-born white persons. TB caused by M. africanum did not show differences in clinical characteristics when compared with TB caused by M. tuberculosis. Clustered cases defined as >2 cases in a county with identical 24-locus mycobacterial interspersed repetitive unit genotypes, were less likely for M. africanum (aOR 0.1, 95% CI 0.1–0.4), which suggests that M. africanum is not commonly transmitted in the United States. PMID:26886258

  13. The Mycobacterium tuberculosis Drugome and Its Polypharmacological Implications

    PubMed Central

    Kinnings, Sarah L.; Xie, Li; Fung, Kingston H.; Jackson, Richard M.; Xie, Lei; Bourne, Philip E.

    2010-01-01

    We report a computational approach that integrates structural bioinformatics, molecular modelling and systems biology to construct a drug-target network on a structural proteome-wide scale. The approach has been applied to the genome of Mycobacterium tuberculosis (M.tb), the causative agent of one of today's most widely spread infectious diseases. The resulting drug-target interaction network for all structurally characterized approved drugs bound to putative M.tb receptors, we refer to as the ‘TB-drugome’. The TB-drugome reveals that approximately one-third of the drugs examined have the potential to be repositioned to treat tuberculosis and that many currently unexploited M.tb receptors may be chemically druggable and could serve as novel anti-tubercular targets. Furthermore, a detailed analysis of the TB-drugome has shed new light on the controversial issues surrounding drug-target networks [1]–[3]. Indeed, our results support the idea that drug-target networks are inherently modular, and further that any observed randomness is mainly caused by biased target coverage. The TB-drugome (http://funsite.sdsc.edu/drugome/TB) has the potential to be a valuable resource in the development of safe and efficient anti-tubercular drugs. More generally the methodology may be applied to other pathogens of interest with results improving as more of their structural proteomes are determined through the continued efforts of structural biology/genomics. PMID:21079673

  14. Meropenem inhibits D,D-carboxypeptidase activity in Mycobacterium tuberculosis.

    PubMed

    Kumar, Pradeep; Arora, Kriti; Lloyd, John R; Lee, Ill Y; Nair, Vinod; Fischer, Elizabeth; Boshoff, Helena I M; Barry, Clifton E

    2012-10-01

    Carbapenems such as meropenem are being investigated for their potential therapeutic utility against highly drug-resistant tuberculosis. These β-lactams target the transpeptidases that introduce interpeptide cross-links into bacterial peptidoglycan thereby controlling rigidity of the bacterial envelope. Treatment of Mycobacterium tuberculosis (Mtb) with the β-lactamase inhibitor clavulanate together with meropenem resulted in rapid, polar, cell lysis releasing cytoplasmic contents. In Mtb it has been previously demonstrated that 3-3 cross-linkages [involving two diaminopimelate (DAP) molecules] predominate over 4-3 cross-linkages (involving one DAP and one D-alanine) in stationary-phase cells. We purified and analysed peptidoglycan from Mtb and found that 3-3 cross-linkages predominate throughout all growth phases and the ratio of 4-3/3-3 linkages does not vary significantly under any growth condition. Meropenem treatment was accompanied by a dramatic accumulation of unlinked pentapeptide stems with no change in the tetrapeptide pools, suggesting that meropenem inhibits both a D,D-carboxypeptidase and an L,D-transpeptidase. We purified a candidate D,D-carboxypeptidase DacB2 and showed that meropenem indeed directly inhibits this enzyme by forming a stable adduct at the enzyme active site. These results suggest that the rapid lysis of meropenem-treated cells is the result of synergistically inhibiting the transpeptidases that introduce 3,3-cross-links while simultaneously limiting the pool of available substrates available for cross-linking. PMID:22906310

  15. Synthesis and evaluation of new 2-aminothiophenes against Mycobacterium tuberculosis.

    PubMed

    Thanna, Sandeep; Knudson, Susan E; Grzegorzewicz, Anna; Kapil, Sunayana; Goins, Christopher M; Ronning, Donald R; Jackson, Mary; Slayden, Richard A; Sucheck, Steven J

    2016-07-01

    Tuberculosis (TB) and its drug resistant forms kills more people than any other infectious disease. This fact emphasizes the need to identify new drugs to treat TB. 2-Aminothiophenes (2AT) have been reported to inhibit Pks13, a validated anti-TB drug target. We synthesized a library of 42 2AT compounds. Among these, compound 33 showed remarkable potency against Mycobacterium tuberculosis (Mtb) H37RV (MIC = 0.23 μM) and showed an impressive potency (MIC = 0.20-0.44 μM) against Mtb strains resistant to isoniazid, rifampicin and fluoroquinolones. The site of action for the compound 33 is presumed to be Pks13 or an earlier enzyme in the mycolic acid biosynthetic pathway. This inference is based on structural similarity of the compound 33 with known Pks13 inhibitors, which is corroborated by mycolic acid biosynthesis studies showing that the compound strongly inhibits the biosynthesis of all forms of mycolic acid in Mtb. In summary, these studies suggest 33 represents a promising anti-TB lead that exhibits activity well below toxicity to human monocytic cells. PMID:27251120

  16. Biochemical Characterization of Uracil Phosphoribosyltransferase from Mycobacterium tuberculosis

    PubMed Central

    Villela, Anne Drumond; Ducati, Rodrigo Gay; Rosado, Leonardo Astolfi; Bloch, Carlos Junior; Prates, Maura Vianna; Gonçalves, Danieli Cristina; Ramos, Carlos Henrique Inacio; Basso, Luiz Augusto; Santos, Diogenes Santiago

    2013-01-01

    Uracil phosphoribosyltransferase (UPRT) catalyzes the conversion of uracil and 5-phosphoribosyl-α-1-pyrophosphate (PRPP) to uridine 5′-monophosphate (UMP) and pyrophosphate (PPi). UPRT plays an important role in the pyrimidine salvage pathway since UMP is a common precursor of all pyrimidine nucleotides. Here we describe cloning, expression and purification to homogeneity of upp-encoded UPRT from Mycobacterium tuberculosis (MtUPRT). Mass spectrometry and N-terminal amino acid sequencing unambiguously identified the homogeneous protein as MtUPRT. Analytical ultracentrifugation showed that native MtUPRT follows a monomer-tetramer association model. MtUPRT is specific for uracil. GTP is not a modulator of MtUPRT ativity. MtUPRT was not significantly activated or inhibited by ATP, UTP, and CTP. Initial velocity and isothermal titration calorimetry studies suggest that catalysis follows a sequential ordered mechanism, in which PRPP binding is followed by uracil, and PPi product is released first followed by UMP. The pH-rate profiles indicated that groups with pK values of 5.7 and 8.1 are important for catalysis, and a group with a pK value of 9.5 is involved in PRPP binding. The results here described provide a solid foundation on which to base upp gene knockout aiming at the development of strategies to prevent tuberculosis. PMID:23424660

  17. Bioluminescence for assessing drug potency against nonreplicating Mycobacterium tuberculosis.

    PubMed

    Vocat, Anthony; Hartkoorn, Ruben C; Lechartier, Benoit; Zhang, Ming; Dhar, Neeraj; Cole, Stewart T; Sala, Claudia

    2015-07-01

    Targeting dormant Mycobacterium tuberculosis represents a challenge to antituberculosis drug discovery programs. We previously reported and validated the use of the streptomycin (STR)-dependent M. tuberculosis 18b strain as a tool for assessing drug potency against nonreplicating bacteria both in vitro and in vivo. In this study, we generated a luminescent 18b strain, named 18b-Lux, by transforming the bacteria with a vector expressing the luxCDABE operon from Photorhabdus luminescens. Luciferase expression was demonstrated under replicating conditions, and, more importantly, luminescence levels significantly above background were detected following STR removal. The sensitivity of STR-starved 18b-Lux to approved and candidate antituberculosis therapeutic agents was evaluated by means of a luciferase assay in a 96-well format. Results mirrored the data obtained with the standard resazurin reduction microplate assay, and the luminescence readout allowed time course assessments of drug efficacy in vitro. Specifically, we proved that bedaquiline, the rifamycins, and sutezolid displayed time-dependent activity against dormant bacteria, while pyrazinamide and SQ109 showed bactericidal effects at the highest concentrations tested. Overall, we established the optimal conditions for an inexpensive, simple, and very sensitive assay with great potential for future applications. PMID:25896710

  18. Bioluminescence for Assessing Drug Potency against Nonreplicating Mycobacterium tuberculosis

    PubMed Central

    Vocat, Anthony; Hartkoorn, Ruben C.; Lechartier, Benoit; Zhang, Ming; Dhar, Neeraj

    2015-01-01

    Targeting dormant Mycobacterium tuberculosis represents a challenge to antituberculosis drug discovery programs. We previously reported and validated the use of the streptomycin (STR)-dependent M. tuberculosis 18b strain as a tool for assessing drug potency against nonreplicating bacteria both in vitro and in vivo. In this study, we generated a luminescent 18b strain, named 18b-Lux, by transforming the bacteria with a vector expressing the luxCDABE operon from Photorhabdus luminescens. Luciferase expression was demonstrated under replicating conditions, and, more importantly, luminescence levels significantly above background were detected following STR removal. The sensitivity of STR-starved 18b-Lux to approved and candidate antituberculosis therapeutic agents was evaluated by means of a luciferase assay in a 96-well format. Results mirrored the data obtained with the standard resazurin reduction microplate assay, and the luminescence readout allowed time course assessments of drug efficacy in vitro. Specifically, we proved that bedaquiline, the rifamycins, and sutezolid displayed time-dependent activity against dormant bacteria, while pyrazinamide and SQ109 showed bactericidal effects at the highest concentrations tested. Overall, we established the optimal conditions for an inexpensive, simple, and very sensitive assay with great potential for future applications. PMID:25896710

  19. Challenging Mycobacterium tuberculosis dormancy mechanisms and their immunodiagnostic potential.

    PubMed

    Chaves, Alexandre Silva; Rodrigues, Michele Fernandes; Mattos, Ana Márcia Menezes; Teixeira, Henrique Couto

    2015-01-01

    Mycobacterium tuberculosis is the etiologic agent of tuberculosis, one of the world's greatest cause of morbidity and mortality due to infectious disease. Many evolutionary mechanisms have contributed to its high level of adaptation as a host pathogen. Prior to become dormant, a group of about 50 genes related to metabolic changes are transcribed by the DosR regulon, one of the most complex and important systems of host-pathogen interaction. This genetic mechanism allows the mycobacteria to persist during long time periods, establishing the so-called latent infection. Even in the presence of a competent immune response, the host cannot eliminate the pathogen, only managing to keep it surrounded by an unfavorable microenvironment for its growth. However, conditions such as immunosuppression may reestablish optimal conditions for bacterial growth, culminating in the onset of active disease. The interactions between the pathogen and its host are still not completely elucidated. Nonetheless, many studies are being carried out in order to clarify this complex relationship, thus creating new possibilities for patient approach and laboratory screening. PMID:26358744

  20. SInCRe—structural interactome computational resource for Mycobacterium tuberculosis

    PubMed Central

    Metri, Rahul; Hariharaputran, Sridhar; Ramakrishnan, Gayatri; Anand, Praveen; Raghavender, Upadhyayula S.; Ochoa-Montaño, Bernardo; Higueruelo, Alicia P.; Sowdhamini, Ramanathan; Chandra, Nagasuma R.; Blundell, Tom L.; Srinivasan, Narayanaswamy

    2015-01-01

    We have developed an integrated database for Mycobacterium tuberculosis H37Rv (Mtb) that collates information on protein sequences, domain assignments, functional annotation and 3D structural information along with protein–protein and protein–small molecule interactions. SInCRe (Structural Interactome Computational Resource) is developed out of CamBan (Cambridge and Bangalore) collaboration. The motivation for development of this database is to provide an integrated platform to allow easily access and interpretation of data and results obtained by all the groups in CamBan in the field of Mtb informatics. In-house algorithms and databases developed independently by various academic groups in CamBan are used to generate Mtb-specific datasets and are integrated in this database to provide a structural dimension to studies on tuberculosis. The SInCRe database readily provides information on identification of functional domains, genome-scale modelling of structures of Mtb proteins and characterization of the small-molecule binding sites within Mtb. The resource also provides structure-based function annotation, information on small-molecule binders including FDA (Food and Drug Administration)-approved drugs, protein–protein interactions (PPIs) and natural compounds that bind to pathogen proteins potentially and result in weakening or elimination of host–pathogen protein–protein interactions. Together they provide prerequisites for identification of off-target binding. Database URL: http://proline.biochem.iisc.ernet.in/sincre PMID:26130660

  1. Genetic markers, genotyping methods & next generation sequencing in Mycobacterium tuberculosis

    PubMed Central

    Desikan, Srinidhi; Narayanan, Sujatha

    2015-01-01

    Molecular epidemiology (ME) is one of the main areas in tuberculosis research which is widely used to study the transmission epidemics and outbreaks of tubercle bacilli. It exploits the presence of various polymorphisms in the genome of the bacteria that can be widely used as genetic markers. Many DNA typing methods apply these genetic markers to differentiate various strains and to study the evolutionary relationships between them. The three widely used genotyping tools to differentiate Mycobacterium tuberculosis strains are IS6110 restriction fragment length polymorphism (RFLP), spacer oligotyping (Spoligotyping), and mycobacterial interspersed repeat units - variable number of tandem repeats (MIRU-VNTR). A new prospect towards ME was introduced with the development of whole genome sequencing (WGS) and the next generation sequencing (NGS) methods, where the entire genome is sequenced that not only helps in pointing out minute differences between the various sequences but also saves time and the cost. NGS is also found to be useful in identifying single nucleotide polymorphisms (SNPs), comparative genomics and also various aspects about transmission dynamics. These techniques enable the identification of mycobacterial strains and also facilitate the study of their phylogenetic and evolutionary traits. PMID:26205019

  2. 2-Phenylindole and Arylsulphonamide: Novel Scaffolds Bactericidal against Mycobacterium tuberculosis

    PubMed Central

    2014-01-01

    A cellular activity-based screen on Mycobacterium tuberculosis (Mtb) H37Rv using a focused library from the AstraZeneca corporate collection led to the identification of 2-phenylindoles and arylsulphonamides, novel antimycobacterial scaffolds. Both the series were bactericidal in vitro and in an intracellular macrophage infection model, active against drug sensitive and drug resistant Mtb clinical isolates, and specific to mycobacteria. The scaffolds showed promising structure–activity relationships; compounds with submicromolar cellular potency were identified during the hit to lead exploration. Furthermore, compounds from both scaffolds were tested for inhibition of known target enzymes or pathways of antimycobacterial drugs including InhA, RNA polymerase, DprE1, topoisomerases, protein synthesis, and oxidative-phosphorylation. Compounds did not inhibit any of the targets suggesting the potential of a possible novel mode of action(s). Hence, both scaffolds provide the opportunity to be developed further as leads and tool compounds to uncover novel mechanisms for tuberculosis drug discovery. PMID:25221657

  3. Clinical value of whole-genome sequencing of Mycobacterium tuberculosis.

    PubMed

    Takiff, Howard E; Feo, Oscar

    2015-09-01

    Whole-genome sequencing (WGS) is now common as a result of new technologies that can rapidly sequence a complete bacterial genome for US$500 or less. Many studies have addressed questions about tuberculosis with WGS, and knowing the sequence of the entire genome, rather than only a few fragments, has greatly increased the precision of molecular epidemiology and contact tracing. Additionally, topics such as the mutation rate, drug resistance, the target of new drugs, and the phylogeny and evolution of the Mycobacterium tuberculosis complex bacteria have been elucidated by WGS. Nonetheless, WGS has not explained differences in transmissibility between strains, or why some strains are more virulent than others or more prone to development of multidrug resistance. With advances in technology, WGS of clinical specimens could become routine in high-income countries; however, its relevance will probably depend on easy to use software to efficiently process the sequences produced and accessible genomic databases that can be mined in future studies. PMID:26277037

  4. Sulfolipid-1 biosynthesis restricts Mycobacterium tuberculosis growth in human macrophages.

    PubMed

    Gilmore, Sarah A; Schelle, Michael W; Holsclaw, Cynthia M; Leigh, Clifton D; Jain, Madhulika; Cox, Jeffery S; Leary, Julie A; Bertozzi, Carolyn R

    2012-05-18

    Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is a highly evolved human pathogen characterized by its formidable cell wall. Many unique lipids and glycolipids from the Mtb cell wall are thought to be virulence factors that mediate host-pathogen interactions. An intriguing example is Sulfolipid-1 (SL-1), a sulfated glycolipid that has been implicated in Mtb pathogenesis, although no direct role for SL-1 in virulence has been established. Previously, we described the biochemical activity of the sulfotransferase Stf0 that initiates SL-1 biosynthesis. Here we show that a stf0-deletion mutant exhibits augmented survival in human but not murine macrophages, suggesting that SL-1 negatively regulates the intracellular growth of Mtb in a species-specific manner. Furthermore, we demonstrate that SL-1 plays a role in mediating the susceptibility of Mtb to a human cationic antimicrobial peptide in vitro, despite being dispensable for maintaining overall cell envelope integrity. Thus, we hypothesize that the species-specific phenotype of the stf0 mutant is reflective of differences in antimycobacterial effector mechanisms of macrophages. PMID:22360425

  5. Spoligotyping for molecular epidemiology of the Mycobacterium tuberculosis complex.

    PubMed

    Driscoll, Jeffrey R

    2009-01-01

    Spacer oligonucleotide typing, or spoligotyping, is a rapid, polymerase chain reaction (PCR)-based method for genotyping strains of the Mycobacterium tuberculosis complex (MTB). Spoligotyping data can be represented in absolute terms (digitally), and the results can be readily shared among laboratories, thereby enabling the creation of large international databases. Since the spoligotype assay was standardized more than 10 yr ago, tens of thousands of isolates have been analyzed, giving a global picture of MTB strain diversity. The method is highly reproducible and has been developed into a high-throughput assay for large molecular epidemiology projects. In the United States, spoligotyping is employed on nearly all newly identified culture-positive cases of tuberculosis as part of a national genotyping program. The strengths of this method include its low cost, its digital data results, the good correlation of its results with other genetics markers, its fair level of overall differentiation of strains, its high-throughput capacity, and its ability to provide species information. However, the method's weaknesses include the inability of spoligotyping to differentiate well within large strain families such as the Beijing family, the potential for convergent evolution of patterns, the limited success in improving the assay through expansion, and the difficulty in obtaining the specialized membranes and instrumentation. PMID:19521871

  6. Succinate dehydrogenase is the regulator of respiration in Mycobacterium tuberculosis.

    PubMed

    Hartman, Travis; Weinrick, Brian; Vilchèze, Catherine; Berney, Michael; Tufariello, Joanne; Cook, Gregory M; Jacobs, William R

    2014-11-01

    In chronic infection, Mycobacterium tuberculosis bacilli are thought to enter a metabolic program that provides sufficient energy for maintenance of the protonmotive force, but is insufficient to meet the demands of cellular growth. We sought to understand this metabolic downshift genetically by targeting succinate dehydrogenase, the enzyme which couples the growth processes controlled by the TCA cycle with the energy production resulting from the electron transport chain. M. tuberculosis contains two operons which are predicted to encode succinate dehydrogenase enzymes (sdh-1 and sdh-2); we found that deletion of Sdh1 contributes to an inability to survive long term stationary phase. Stable isotope labeling and mass spectrometry revealed that Sdh1 functions as a succinate dehydrogenase during aerobic growth, and that Sdh2 is dispensable for this catalysis, but partially overlapping activities ensure that the loss of one enzyme can incompletely compensate for loss of the other. Deletion of Sdh1 disturbs the rate of respiration via the mycobacterial electron transport chain, resulting in an increased proportion of reduced electron carrier (menaquinol) which leads to increased oxygen consumption. The loss of respiratory control leads to an inability to recover from stationary phase. We propose a model in which succinate dehydrogenase is a governor of cellular respiration in the adaptation to low oxygen environments. PMID:25412183

  7. Evaluation of an Immunochromatographic Assay Kit for Rapid Identification of Mycobacterium tuberculosis Complex in Clinical Isolates▿

    PubMed Central

    Park, Mi Young; Kim, Young Jin; Hwang, Sang Hyun; Kim, Hyoung Hoi; Lee, Eun Yup; Jeong, Seok Hoon; Chang, Chulhun L.

    2009-01-01

    We evaluated a new immunochromatographic assay (ICA) using mouse monoclonal anti-MPT64 antibody for rapid discrimination between Mycobacterium tuberculosis and nontuberculous mycobacteria in clinical isolates. A study with mycobacteria and other organisms showed excellent sensitivity (≅99%) and specificity (100%) and an appropriate detection limit (105 CFU/ml) when tested with M. tuberculosis H37Rv. This ICA can simplify the identification of M. tuberculosis in clinical laboratories. PMID:19052177

  8. Mycobacterium tuberculosis TlyA Protein Negatively Regulates T Helper (Th) 1 and Th17 Differentiation and Promotes Tuberculosis Pathogenesis*

    PubMed Central

    Rahman, Md. Aejazur; Sobia, Parveen; Dwivedi, Ved Prakash; Bhawsar, Aakansha; Singh, Dhiraj Kumar; Sharma, Pawan; Moodley, Prashini; Van Kaer, Luc; Bishai, William R; Das, Gobardhan

    2015-01-01

    Mycobacterium tuberculosis, the causative agent of tuberculosis, is an ancient pathogen and a major cause of death worldwide. Although various virulence factors of M. tuberculosis have been identified, its pathogenesis remains incompletely understood. TlyA is a virulence factor in several bacterial infections and is evolutionarily conserved in many Gram-positive bacteria, but its function in M. tuberculosis pathogenesis has not been elucidated. Here, we report that TlyA significantly contributes to the pathogenesis of M. tuberculosis. We show that a TlyA mutant M. tuberculosis strain induces increased IL-12 and reduced IL-1β and IL-10 cytokine responses, which sharply contrasts with the immune responses induced by wild type M. tuberculosis. Furthermore, compared with wild type M. tuberculosis, TlyA-deficient M. tuberculosis bacteria are more susceptible to autophagy in macrophages. Consequently, animals infected with the TlyA mutant M. tuberculosis organisms exhibited increased host-protective immune responses, reduced bacillary load, and increased survival compared with animals infected with wild type M. tuberculosis. Thus, M. tuberculosis employs TlyA as a host evasion factor, thereby contributing to its virulence. PMID:25847237

  9. Mycobacterium africanum—Review of an Important Cause of Human Tuberculosis in West Africa

    PubMed Central

    de Jong, Bouke C.; Antonio, Martin; Gagneux, Sebastien

    2010-01-01

    Mycobacterium africanum consists of two phylogenetically distinct lineages within the Mycobacterium tuberculosis complex, known as M. africanum West African 1 and M. africanum West African 2. These lineages are restricted to West Africa, where they cause up to half of human pulmonary tuberculosis. In this review we discuss the definition of M. africanum, describe the prevalence and restricted geographical distribution of M. africanum West African 1 and 2, review the occurrence of M. africanum in animals, and summarize the phenotypic differences described thus far between M. africanum and M. tuberculosis sensu stricto. PMID:20927191

  10. Immunogenicity and protective efficacy of novel Mycobacterium tuberculosis antigens.

    PubMed

    Derrick, Steven C; Yabe, Idalia M; Yang, Amy; Kolibab, Kristopher; Hollingsworth, Brynn; Kurtz, Sherry L; Morris, Sheldon

    2013-09-23

    With tuberculosis continuing to be a major cause of global morbidity and mortality, a new vaccine is urgently needed. Tuberculosis subunit vaccines have been shown to induce robust immune responses in humans and are a potentially safer alternative to BCG for use in HIV-endemic areas. In this study, we investigated the protective efficacy of 16 different novel Mycobacterium tuberculosis antigens using an aerogenic mouse model of pulmonary tuberculosis. These antigens were tested as subunit vaccines formulated in dimethyl dioctadecyl ammonium bromide (DDA) - D(+) with trehalose 6,6 dibenenate (TDB) (DDA/TDB) adjuvant administered alone as monovalent vaccines or in combination. Six of these antigens (Rv1626, Rv1735, Rv1789, Rv2032, Rv2220, and Rv3478) were shown to consistently and significantly reduce bacterial burdens in the lungs of mice relative to nonvaccinated controls. Three of these six (Rv1789, Rv2220, and Rv3478) induced levels of protective immunity that were essentially equivalent to protection induced by the highly immunogenic antigen 85B (>0.5 log₁₀CFU reduction in the lungs relative to naïve mice). Importantly, when these three antigens were combined, protection essentially equivalent to that mediated by BCG was observed. When either Rv1626 or Rv2032 were combined with the highly protective E6-85 fusion protein (antigen 85B fused to ESAT-6), the protection observed was equivalent to BCG-induced protection at one and three months post-aerosol infection and was significantly greater than the protection observed when E6-85 was administered alone at 3 months post-infection. Using multiparameter flow cytometry, monofunctional IFNγ CD4T cells and different multifunctional CD4T cell subsets capable of secreting multiple cytokines (IFNγ, TNFα and/or IL-2) were shown to be induced by the three most protective antigens with splenocyte CD4T cell frequencies significantly greater than observed in naïve controls. The identification of these highly

  11. Comparative Proteomic Analysis of Aminoglycosides Resistant and Susceptible Mycobacterium tuberculosis Clinical Isolates for Exploring Potential Drug Targets

    PubMed Central

    Sharma, Divakar; Kumar, Bhavnesh; Lata, Manju; Joshi, Beenu; Venkatesan, Krishnamurthy; Shukla, Sangeeta; Bisht, Deepa

    2015-01-01

    Aminoglycosides, amikacin (AK) and kanamycin (KM) are second line anti-tuberculosis drugs used to treat tuberculosis (TB) and resistance to them affects the treatment. Membrane and membrane associated proteins have an anticipated role in biological processes and pathogenesis and are potential targets for the development of new diagnostics/vaccine/therapeutics. In this study we compared membrane and membrane associated proteins of AK and KM resistant and susceptible Mycobacterium tuberculosis isolates by 2DE coupled with MALDI-TOF/TOF-MS and bioinformatic tools. Twelve proteins were found to have increased intensities (PDQuest Advanced Software) in resistant isolates and were identified as ATP synthase subunit alpha (Rv1308), Trigger factor (Rv2462c), Dihydrolipoyl dehydrogenase (Rv0462), Elongation factor Tu (Rv0685), Transcriptional regulator MoxR1(Rv1479), Universal stress protein (Rv2005c), 35kDa hypothetical protein (Rv2744c), Proteasome subunit alpha (Rv2109c), Putative short-chain type dehydrogenase/reductase (Rv0148), Bacterioferritin (Rv1876), Ferritin (Rv3841) and Alpha-crystallin/HspX (Rv2031c). Among these Rv2005c, Rv2744c and Rv0148 are proteins with unknown functions. Docking showed that both drugs bind to the conserved domain (Usp, PspA and SDR domain) of these hypothetical proteins and GPS-PUP predicted potential pupylation sites within them. Increased intensities of these proteins and proteasome subunit alpha might not only be neutralized/modulated the drug molecules but also involved in protein turnover to overcome the AK and KM resistance. Besides that Rv1876, Rv3841 and Rv0685 were found to be associated with iron regulation signifying the role of iron in resistance. Further research is needed to explore how these potential protein targets contribute to resistance of AK and KM. PMID:26436944

  12. Structural and functional studies of phosphoenolpyruvate carboxykinase from Mycobacterium tuberculosis.

    PubMed

    Machová, Iva; Snášel, Jan; Dostál, Jiří; Brynda, Jiří; Fanfrlík, Jindřich; Singh, Mahavir; Tarábek, Ján; Vaněk, Ondřej; Bednárová, Lucie; Pichová, Iva

    2015-01-01

    Tuberculosis, the second leading infectious disease killer after HIV, remains a top public health priority. The causative agent of tuberculosis, Mycobacterium tuberculosis (Mtb), which can cause both acute and clinically latent infections, reprograms metabolism in response to the host niche. Phosphoenolpyruvate carboxykinase (Pck) is the enzyme at the center of the phosphoenolpyruvate-pyruvate-oxaloacetate node, which is involved in regulating the carbon flow distribution to catabolism, anabolism, or respiration in different states of Mtb infection. Under standard growth conditions, Mtb Pck is associated with gluconeogenesis and catalyzes the metal-dependent formation of phosphoenolpyruvate. In non-replicating Mtb, Pck can catalyze anaplerotic biosynthesis of oxaloacetate. Here, we present insights into the regulation of Mtb Pck activity by divalent cations. Through analysis of the X-ray structure of Pck-GDP and Pck-GDP-Mn2+ complexes, mutational analysis of the GDP binding site, and quantum mechanical (QM)-based analysis, we explored the structural determinants of efficient Mtb Pck catalysis. We demonstrate that Mtb Pck requires presence of Mn2+ and Mg2+ cations for efficient catalysis of gluconeogenic and anaplerotic reactions. The anaplerotic reaction, which preferably functions in reducing conditions that are characteristic for slowed or stopped Mtb replication, is also effectively activated by Fe2+ in the presence of Mn2+ or Mg2+ cations. In contrast, simultaneous presence of Fe2+ and Mn2+ or Mg2+ inhibits the gluconeogenic reaction. These results suggest that inorganic ions can contribute to regulation of central carbon metabolism by influencing the activity of Pck. Furthermore, the X-ray structure determination, biochemical characterization, and QM analysis of Pck mutants confirmed the important role of the Phe triad for proper binding of the GDP-Mn2+ complex in the nucleotide binding site and efficient catalysis of the anaplerotic reaction. PMID:25798914

  13. Portrait of a Pathogen: The Mycobacterium tuberculosis Proteome In Vivo

    PubMed Central

    Kruh, Nicole A.; Troudt, Jolynn; Izzo, Angelo; Prenni, Jessica; Dobos, Karen M.

    2010-01-01

    Background Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is a facultative intracellular pathogen that can persist within the host. The bacteria are thought to be in a state of reduced replication and metabolism as part of the chronic lung infection. Many in vitro studies have dissected the hypothesized environment within the infected lung, defining the bacterial response to pH, starvation and hypoxia. While these experiments have afforded great insight, the picture remains incomplete. The only way to study the combined effects of these environmental factors and the mycobacterial response is to study the bacterial response in vivo. Methodology/Principal Findings We used the guinea pig model of tuberculosis to examine the bacterial proteome during the early and chronic stages of disease. Lungs were harvested thirty and ninety days after aerosol challenge with Mtb, and analyzed by liquid chromatography-mass spectrometry. To date, in vivo proteomics of the tubercle bacillus has not been described and this work has generated the first large-scale shotgun proteomic data set, comprising over 500 unique protein identifications. Cell wall and cell wall processes, and intermediary metabolism and respiration were the two major functional classes of proteins represented in the infected lung. These classes of proteins displayed the greatest heterogeneity indicating important biological processes for establishment of a productive bacterial infection and its persistence. Proteins necessary for adaptation throughout infection, such as nitrate/nitrite reduction were found at both time points. The PE-PPE protein class, while not well characterized, represented the third most abundant category and showed the most consistent expression during the infection. Conclusions/Significance Cumulatively, the results of this work may provide the basis for rational drug design – identifying numerous Mtb proteins, from essential kinases to products involved in

  14. Construction, molecular modeling, and simulation of Mycobacterium tuberculosis cell walls.

    PubMed

    Hong, Xuan; Hopfinger, A J

    2004-01-01

    The mycobacterial cell wall is extraordinarily thick and tight consisting mainly of (1). long chain fatty acids, the mycolic acids, and (2). a unique polysaccharide, arabinogalactan (AG). These two chemical constituents are covalently linked through ester bonds. Minnikin (The Biology of the Mycobacteria; Academic: London, 1982) proposed that the mycobacterial cell wall is composed of an asymmetric lipid bilayer. The inner leaflet of the cell wall contains mycolic acids covalently linked to AG. This inner leaflet is believed to have the lowest permeability to organic compounds of the overall cell wall. Conformational search and molecular dynamics simulation were used to explore the conformational profile of AG and the conformations and structural organization of the mycolic acid-AG complex, and overall, an inner leaflet molecular model of the cell wall was constructed. The terminal arabinose residues of AG that serve as linkers between AG and mycolic acids were found to exist in four major chemical configurations. The mycolate hydrocarbon chains were determined to be tightly packed and perpendicular to the "plane" formed by the oxygen atoms of the 5-hydroxyl groups of the terminal arabinose residues. For Mycobacterium tuberculosis, the average packing distance between mycolic acids is estimated to be approximately 7.3 A. Thus, Minnikin's model is supported by this computational study. Overall, this modeling and simulation approach provides a way to probe the mechanism of low permeability of the cell wall and the intrinsic drug resistance of M. tuberculosis. In addition, monolayer models were built for both dipalmitoylphosphatidylethanolamine and dimyristoylphosphatidylcholine, two common phospholipids in bacterial and animal membranes, respectively. Structural comparisons of these cell wall phospholipid membrane models were made to the M. tuberculosis cell wall model. PMID:15132700

  15. Structural and Functional Studies of Phosphoenolpyruvate Carboxykinase from Mycobacterium tuberculosis

    PubMed Central

    Machová, Iva; Snášel, Jan; Dostál, Jiří; Brynda, Jiří; Fanfrlík, Jindřich; Singh, Mahavir; Tarábek, Ján; Vaněk, Ondřej; Bednárová, Lucie; Pichová, Iva

    2015-01-01

    Tuberculosis, the second leading infectious disease killer after HIV, remains a top public health priority. The causative agent of tuberculosis, Mycobacterium tuberculosis (Mtb), which can cause both acute and clinically latent infections, reprograms metabolism in response to the host niche. Phosphoenolpyruvate carboxykinase (Pck) is the enzyme at the center of the phosphoenolpyruvate-pyruvate-oxaloacetate node, which is involved in regulating the carbon flow distribution to catabolism, anabolism, or respiration in different states of Mtb infection. Under standard growth conditions, Mtb Pck is associated with gluconeogenesis and catalyzes the metal-dependent formation of phosphoenolpyruvate. In non-replicating Mtb, Pck can catalyze anaplerotic biosynthesis of oxaloacetate. Here, we present insights into the regulation of Mtb Pck activity by divalent cations. Through analysis of the X-ray structure of Pck-GDP and Pck-GDP-Mn2+ complexes, mutational analysis of the GDP binding site, and quantum mechanical (QM)-based analysis, we explored the structural determinants of efficient Mtb Pck catalysis. We demonstrate that Mtb Pck requires presence of Mn2+ and Mg2+ cations for efficient catalysis of gluconeogenic and anaplerotic reactions. The anaplerotic reaction, which preferably functions in reducing conditions that are characteristic for slowed or stopped Mtb replication, is also effectively activated by Fe2+ in the presence of Mn2+ or Mg2+ cations. In contrast, simultaneous presence of Fe2+ and Mn2+ or Mg2+ inhibits the gluconeogenic reaction. These results suggest that inorganic ions can contribute to regulation of central carbon metabolism by influencing the activity of Pck. Furthermore, the X-ray structure determination, biochemical characterization, and QM analysis of Pck mutants confirmed the important role of the Phe triad for proper binding of the GDP-Mn2+ complex in the nucleotide binding site and efficient catalysis of the anaplerotic reaction. PMID:25798914

  16. Micrococcin P1 - A bactericidal thiopeptide active against Mycobacterium tuberculosis.

    PubMed

    Degiacomi, Giulia; Personne, Yoann; Mondésert, Guillaume; Ge, Xueliang; Mandava, Chandra Sekhar; Hartkoorn, Ruben C; Boldrin, Francesca; Goel, Pavitra; Peisker, Kristin; Benjak, Andrej; Barrio, Maria Belén; Ventura, Marcello; Brown, Amanda C; Leblanc, Véronique; Bauer, Armin; Sanyal, Suparna; Cole, Stewart T; Lagrange, Sophie; Parish, Tanya; Manganelli, Riccardo

    2016-09-01

    The lack of proper treatment for serious infectious diseases due to the emergence of multidrug resistance reinforces the need for the discovery of novel antibiotics. This is particularly true for tuberculosis (TB) for which 3.7% of new cases and 20% of previously treated cases are estimated to be caused by multi-drug resistant strains. In addition, in the case of TB, which claimed 1.5 million lives in 2014, the treatment of the least complicated, drug sensitive cases is lengthy and disagreeable. Therefore, new drugs with novel targets are urgently needed to control resistant Mycobacterium tuberculosis strains. In this manuscript we report the characterization of the thiopeptide micrococcin P1 as an anti-tubercular agent. Our biochemical experiments show that this antibiotic inhibits the elongation step of protein synthesis in mycobacteria. We have further identified micrococcin resistant mutations in the ribosomal protein L11 (RplK); the mutations were located in the proline loop at the N-terminus. Reintroduction of the mutations into a clean genetic background, confirmed that they conferred resistance, while introduction of the wild type RplK allele into resistant strains re-established sensitivity. We also identified a mutation in the 23S rRNA gene. These data, in good agreement with previous structural studies suggest that also in M. tuberculosis micrococcin P1 functions by binding to the cleft between the 23S rRNA and the L11 protein loop, thus interfering with the binding of elongation factors Tu and G (EF-Tu and EF-G) and inhibiting protein translocation. PMID:27553416

  17. Diagnostic value of antibody responses to multiple antigens from Mycobacterium tuberculosis in active and latent tuberculosis.

    PubMed

    Senoputra, Muhammad Andrian; Shiratori, Beata; Hasibuan, Fakhrial Mirwan; Koesoemadinata, Raspati Cundarani; Apriani, Lika; Ashino, Yugo; Ono, Kenji; Oda, Tetsuya; Matsumoto, Makoto; Suzuki, Yasuhiko; Alisjahbana, Bachti; Hattori, Toshio

    2015-11-01

    We investigated the antibody responses to 10 prospective Mycobacterium tuberculosis (MTB) antigens and evaluated their ability to discriminate between latent (LTBI) and active pulmonary tuberculosis (TB). Our results indicate that plasma levels of anti-α-crystallin (ACR), antilipoarabinomannan, anti-trehalose 6,6'-dimycolate, and anti-tubercular-glycolipid antigen antibodies were higher in patients with active TB, compared to those in the LTBI and control subjects. No differences in the antibodies were observed between the control and LTBI subjects. Antibodies against the glycolipid antigens could not distinguish between Mycobacterium avium complex (MAC)-negative TB patients and MAC-infected LTBI individuals. The most useful serological marker was antibodies to ACR, with MAC-negative TB patients having higher titers than those observed in MAC-positive LTBI and control subjects. Our data indicate that antibody to ACR is a promising target for the serological diagnosis of patients with active TB patients. When dealing with antiglycolipid antibodies, MAC coinfection should always be considered in serological studies. PMID:26307672

  18. Isolation of Mycobacterium kumamotonense from a patient with pulmonary infection and latent tuberculosis.

    PubMed

    Kontos, Fanourios; Mavromanolakis, Dimitrios Nikitas; Zande, Marina Chari; Gitti, Zoe Georgios

    2016-01-01

    Mycobacterium kumamotonense is a novel, slow-growing non-chromogenic nontuberculous mycobacterium, which belongs to Mycobacterium terrae complex. We report, for the first time in Greece, the isolation of M. kumamotonense from an immunocompetent patient with pulmonary infection and latent tuberculosis. M. kumamotonense was identified by sequencing analysis of 16S rDNA and 65-kDa heat shock protein genes while by commercial molecular assays it was misidentified as Mycobacterium celatum. Antibiotic susceptibility testing was performed by the reference broth microdilution method. The strain was susceptible to amikacin, clarithromycin, rifampin, ciprofloxacin, moxifloxacin, rifabutin, ethambutol and linezolid. PMID:27080783

  19. Identification of gene targets against dormant phase Mycobacterium tuberculosis infections

    PubMed Central

    Murphy, Dennis J; Brown, James R

    2007-01-01

    Background Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), infects approximately 2 billion people worldwide and is the leading cause of mortality due to infectious disease. Current TB therapy involves a regimen of four antibiotics taken over a six month period. Patient compliance, cost of drugs and increasing incidence of drug resistant M. tuberculosis strains have added urgency to the development of novel TB therapies. Eradication of TB is affected by the ability of the bacterium to survive up to decades in a dormant state primarily in hypoxic granulomas in the lung and to cause recurrent infections. Methods The availability of M. tuberculosis genome-wide DNA microarrays has lead to the publication of several gene expression studies under simulated dormancy conditions. However, no single model best replicates the conditions of human pathogenicity. In order to identify novel TB drug targets, we performed a meta-analysis of multiple published datasets from gene expression DNA microarray experiments that modeled infection leading to and including the dormant state, along with data from genome-wide insertional mutagenesis that examined gene essentiality. Results Based on the analysis of these data sets following normalization, several genome wide trends were identified and used to guide the selection of targets for therapeutic development. The trends included the significant up-regulation of genes controlled by devR, down-regulation of protein and ATP synthesis, and the adaptation of two-carbon metabolism to the hypoxic and nutrient limited environment of the granuloma. Promising targets for drug discovery were several regulatory elements (devR/devS, relA, mprAB), enzymes involved in redox balance and respiration, sulfur transport and fixation, pantothenate, isoprene, and NAD biosynthesis. The advantages and liabilities of each target are discussed in the context of enzymology, bacterial pathways, target tractability, and drug development

  20. The Guinea-Bissau family of Mycobacterium tuberculosis complex revisited.

    PubMed

    Groenheit, Ramona; Ghebremichael, Solomon; Svensson, Jenny; Rabna, Paulo; Colombatti, Raffaella; Riccardi, Fabio; Couvin, David; Hill, Véronique; Rastogi, Nalin; Koivula, Tuija; Källenius, Gunilla

    2011-01-01

    The Guinea-Bissau family of strains is a unique group of the Mycobacterium tuberculosis complex that, although genotypically closely related, phenotypically demonstrates considerable heterogeneity. We have investigated 414 M. tuberculosis complex strains collected in Guinea-Bissau between 1989 and 2008 in order to further characterize the Guinea-Bissau family of strains. To determine the strain lineages present in the study sample, binary outcomes of spoligotyping were compared with spoligotypes existing in the international database SITVIT2. The major circulating M. tuberculosis clades ranked in the following order: AFRI (n = 195, 47.10%), Latin-American-Mediterranean (LAM) (n = 75, 18.12%), ill-defined T clade (n = 53, 12.8%), Haarlem (n = 37, 8.85%), East-African-Indian (EAI) (n = 25, 6.04%), Unknown (n = 12, 2.87%), Beijing (n = 7, 1.68%), X clade (n = 4, 0.96%), Manu (n = 4, 0.97%), CAS (n = 2, 0.48%). Two strains of the LAM clade isolated in 2007 belonged to the Cameroon family (SIT61). All AFRI isolates except one belonged to the Guinea-Bissau family, i.e. they have an AFRI_1 spoligotype pattern, they have a distinct RFLP pattern with low numbers of IS6110 insertions, and they lack the regions of difference RD7, RD8, RD9 and RD10, RD701 and RD702. This profile classifies the Guinea-Bissau family, irrespective of phenotypic biovar, as part of the M. africanum West African 2 lineage, or the AFRI_1 sublineage according to the spoligtyping nomenclature. Guinea-Bissau family strains display a variation of biochemical traits classically used to differentiate M. tuberculosis from M. bovis. Yet, the differential expression of these biochemical traits was not related to any genes so far investigated (narGHJI and pncA). Guinea-Bissau has the highest prevalence of M. africanum recorded in the African continent, and the Guinea-Bissau family shows a high phylogeographical specificity for Western Africa, with Guinea-Bissau being the

  1. Hemophagocytic lymphohistiocytosis: An unusual complication in disseminated Mycobacterium tuberculosis

    PubMed Central

    Padhi, Somanath; Ravichandran, Kandasamy; Sahoo, Jayaprakash; Varghese, Renu G’Boy; Basheer, Aneesh

    2015-01-01

    Background: Hemophagocytic lymphohistiocytosis (HLH) is an uncommon, potentially fatal, hyperinflammatory syndrome that may rarely complicate the clinical course of disseminated Mycobacterium tuberculosis (MTB). The clinical course of tuberculosis-associated HLH (TB-HLH) has been reported to be unpredictable. Materials and Methods: Here we describe the clinicopathological features, laboratory parameters, management, and outcome data of a patient who satisfied the 2004 diagnostic criteria for HLH secondary to disseminated MTB; we also do a systematic review of the international literature on TB-HLH. The literature review (January 1975–March 2014) found that HLH complicated the clinical course of 63 tuberculosis patients (41 males, 22 females, mean age = 45 ± 23.5 years) with a high mortality rate of 49% (31/63 died). The mean serum ferritin level (n = 44/63) was 5963 ng/mL (range 500–38,539 ng/mL); and a higher proportion (54.2%) of patients had pancytopenia at presentation. On univariate analysis (n = 53/63), age >30 years [hazard ratio (HR): 2.79, 95% confidence interval (CI):1.03–7.56, P = 0.03], presence of comorbidities (HR 4.59, CI: 1.08–19.52, P = 0.04), marked hemophagocytosis in bone marrow (HR: 2.65, CI: 1.16–6.05, P = 0.02), and nonusage/delayed usage of antitubercular therapy (ATT) (HR: 3.44, CI: 1.51–7.87, P = 0.003) were associated with decreased survival, though none of these parameters attained statistical significance (P > 0.05) in multivariate analysis. Usage of corticosteroids and/or immunomodulator drugs (HR 1.00, CI: 0.66–3.22, P = 0.35) did not alter the outcome in these patients. Conclusion: HLH should be considered as a differential diagnosis in patients with tuberculosis who present with cytopenias, organomegaly, and coagulopathy. Strong clinical suspicion and early usage of ATT might be useful in reducing the morbidity and mortality. The utility of immunosuppressive/immunomodulator therapy lacks general concensus among

  2. Detection of Mycobacterium tuberculosis and Mycobacterium avium Complexes by Real-Time PCR in Bovine Milk from Brazilian Dairy Farms.

    PubMed

    Bezerra, André Vinícius Andrade; Dos Reis, Emily Marques; Rodrigues, Rogério Oliveira; Cenci, Alexander; Cerva, Cristine; Mayer, Fabiana Quoos

    2015-05-01

    Foodborne diseases are a public health problem worldwide. The consumption of contaminated raw milk has been recognized as a major cause of transmission of bovine tuberculosis to humans. Other mycobacteria that may be present in raw milk and may cause diseases are those belonging to the Mycobacterium avium complex. In this study, molecular biology tools were applied to investigate raw milk contamination with Mycobacterium spp. in family dairy farms from Rio Grande do Sul, southern Brazil. Furthermore, different variables related to the source of the milk, herd characteristics, and management were evaluated for their effect on milk contamination. Five hundred and two samples were analyzed, of which 354 were from the Northwest region (102 farms with samples from 93 bulk tanks and 261 animals) and 148 from the South region of the state (22 farms with samples from 23 bulk tanks and 125 animals). Among them, 10 (1.99%) and 7 (1.39%) were positive for Mycobacterium tuberculosis (9 confirmed as Mycobacterium bovis) and M. avium complexes, respectively. There was no difference in the frequencies of positive samples between the regions or the sample sources. Of the positive samples, 4 were collected from a bulk tank (1 positive for M. avium and 3 for M. tuberculosis). Moreover, 1 sample was positive concomitantly for M. tuberculosis and M. avium complexes. On risk analysis, no variable was associated with raw milk contamination by M. tuberculosis complex species. However, washing the udders of all animals and drying them with paper towels were weakly classified as risk factors for M. avium contamination. Positive samples were obtained from both animals and bulk tanks, which emphasizes the importance of tuberculosis control programs and provides evidence that milk monitoring can be used as a control practice. Moreover, the findings of this study reinforce the need for awareness of the problems of raw milk consumption among the general population. PMID:25951404

  3. Cloning and sequence analysis of a class A beta-lactamase from Mycobacterium tuberculosis H37Ra.

    PubMed Central

    Hackbarth, C J; Unsal, I; Chambers, H F

    1997-01-01

    A cosmid library from Mycobacterium tuberculosis H37Ra was introduced into Mycobacterium smegmatis, and eight recombinant clones with increased resistance to cefoxitin were identified. Isoelectric focusing detected an M. tuberculosis-derived beta-lactamase in one of these recombinant clones. A sequence analysis identified it as a class A beta-lactamase whose expression correlated with the increased resistance phenotype. PMID:9145897

  4. Mycobacterium bovis infection of cattle and white-tailed deer: Translational research of relevance to human tuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tuberculosis (TB) is a premier example of a disease complex with pathogens primarily affecting humans (i.e., Mycobacterium tuberculosis) or livestock and wildlife (i.e., Mycobacterium bovis) and with a long history of inclusive collaborations between physicians and veterinarians. Advances with the s...

  5. Management of latent Mycobacterium tuberculosis infection: WHO guidelines for low tuberculosis burden countries.

    PubMed

    Getahun, Haileyesus; Matteelli, Alberto; Abubakar, Ibrahim; Aziz, Mohamed Abdel; Baddeley, Annabel; Barreira, Draurio; Den Boon, Saskia; Borroto Gutierrez, Susana Marta; Bruchfeld, Judith; Burhan, Erlina; Cavalcante, Solange; Cedillos, Rolando; Chaisson, Richard; Chee, Cynthia Bin-Eng; Chesire, Lucy; Corbett, Elizabeth; Dara, Masoud; Denholm, Justin; de Vries, Gerard; Falzon, Dennis; Ford, Nathan; Gale-Rowe, Margaret; Gilpin, Chris; Girardi, Enrico; Go, Un-Yeong; Govindasamy, Darshini; D Grant, Alison; Grzemska, Malgorzata; Harris, Ross; Horsburgh, C Robert; Ismayilov, Asker; Jaramillo, Ernesto; Kik, Sandra; Kranzer, Katharina; Lienhardt, Christian; LoBue, Philip; Lönnroth, Knut; Marks, Guy; Menzies, Dick; Migliori, Giovanni Battista; Mosca, Davide; Mukadi, Ya Diul; Mwinga, Alwyn; Nelson, Lisa; Nishikiori, Nobuyuki; Oordt-Speets, Anouk; Rangaka, Molebogeng Xheedha; Reis, Andreas; Rotz, Lisa; Sandgren, Andreas; Sañé Schepisi, Monica; Schünemann, Holger J; Sharma, Surender Kumar; Sotgiu, Giovanni; Stagg, Helen R; Sterling, Timothy R; Tayeb, Tamara; Uplekar, Mukund; van der Werf, Marieke J; Vandevelde, Wim; van Kessel, Femke; van't Hoog, Anna; Varma, Jay K; Vezhnina, Natalia; Voniatis, Constantia; Vonk Noordegraaf-Schouten, Marije; Weil, Diana; Weyer, Karin; Wilkinson, Robert John; Yoshiyama, Takashi; Zellweger, Jean Pierre; Raviglione, Mario

    2015-12-01

    Latent tuberculosis infection (LTBI) is characterised by the presence of immune responses to previously acquired Mycobacterium tuberculosis infection without clinical evidence of active tuberculosis (TB). Here we report evidence-based guidelines from the World Health Organization for a public health approach to the management of LTBI in high risk individuals in countries with high or middle upper income and TB incidence of <100 per 100 000 per year. The guidelines strongly recommend systematic testing and treatment of LTBI in people living with HIV, adult and child contacts of pulmonary TB cases, patients initiating anti-tumour necrosis factor treatment, patients receiving dialysis, patients preparing for organ or haematological transplantation, and patients with silicosis. In prisoners, healthcare workers, immigrants from high TB burden countries, homeless persons and illicit drug users, systematic testing and treatment of LTBI is conditionally recommended, according to TB epidemiology and resource availability. Either commercial interferon-gamma release assays or Mantoux tuberculin skin testing could be used to test for LTBI. Chest radiography should be performed before LTBI treatment to rule out active TB disease. Recommended treatment regimens for LTBI include: 6 or 9 month isoniazid; 12 week rifapentine plus isoniazid; 3-4 month isoniazid plus rifampicin; or 3-4 month rifampicin alone. PMID:26405286

  6. Management of latent Mycobacterium tuberculosis infection: WHO guidelines for low tuberculosis burden countries

    PubMed Central

    Matteelli, Alberto; Abubakar, Ibrahim; Aziz, Mohamed Abdel; Baddeley, Annabel; Barreira, Draurio; Den Boon, Saskia; Borroto Gutierrez, Susana Marta; Bruchfeld, Judith; Burhan, Erlina; Cavalcante, Solange; Cedillos, Rolando; Chaisson, Richard; Chee, Cynthia Bin-Eng; Chesire, Lucy; Corbett, Elizabeth; Dara, Masoud; Denholm, Justin; de Vries, Gerard; Falzon, Dennis; Ford, Nathan; Gale-Rowe, Margaret; Gilpin, Chris; Girardi, Enrico; Go, Un-Yeong; Govindasamy, Darshini; D. Grant, Alison; Grzemska, Malgorzata; Harris, Ross; Horsburgh Jr, C. Robert; Ismayilov, Asker; Jaramillo, Ernesto; Kik, Sandra; Kranzer, Katharina; Lienhardt, Christian; LoBue, Philip; Lönnroth, Knut; Marks, Guy; Menzies, Dick; Migliori, Giovanni Battista; Mosca, Davide; Mukadi, Ya Diul; Mwinga, Alwyn; Nelson, Lisa; Nishikiori, Nobuyuki; Oordt-Speets, Anouk; Rangaka, Molebogeng Xheedha; Reis, Andreas; Rotz, Lisa; Sandgren, Andreas; Sañé Schepisi, Monica; Schünemann, Holger J.; Sharma, Surender Kumar; Sotgiu, Giovanni; Stagg, Helen R.; Sterling, Timothy R.; Tayeb, Tamara; Uplekar, Mukund; van der Werf, Marieke J.; Vandevelde, Wim; van Kessel, Femke; van't Hoog, Anna; Varma, Jay K.; Vezhnina, Natalia; Voniatis, Constantia; Vonk Noordegraaf-Schouten, Marije; Weil, Diana; Weyer, Karin; Wilkinson, Robert John; Yoshiyama, Takashi; Zellweger, Jean Pierre; Raviglione, Mario

    2015-01-01

    Latent tuberculosis infection (LTBI) is characterised by the presence of immune responses to previously acquired Mycobacterium tuberculosis infection without clinical evidence of active tuberculosis (TB). Here we report evidence-based guidelines from the World Health Organization for a public health approach to the management of LTBI in high risk individuals in countries with high or middle upper income and TB incidence of <100 per 100 000 per year. The guidelines strongly recommend systematic testing and treatment of LTBI in people living with HIV, adult and child contacts of pulmonary TB cases, patients initiating anti-tumour necrosis factor treatment, patients receiving dialysis, patients preparing for organ or haematological transplantation, and patients with silicosis. In prisoners, healthcare workers, immigrants from high TB burden countries, homeless persons and illicit drug users, systematic testing and treatment of LTBI is conditionally recommended, according to TB epidemiology and resource availability. Either commercial interferon-gamma release assays or Mantoux tuberculin skin testing could be used to test for LTBI. Chest radiography should be performed before LTBI treatment to rule out active TB disease. Recommended treatment regimens for LTBI include: 6 or 9 month isoniazid; 12 week rifapentine plus isoniazid; 3–4 month isoniazid plus rifampicin; or 3–4 month rifampicin alone. PMID:26405286

  7. Molecular diversity of Mycobacterium tuberculosis isolates from patients with pulmonary tuberculosis in Mozambique

    PubMed Central

    2010-01-01

    Background Mozambique is one of the countries with the highest burden of tuberculosis (TB) in Sub-Saharan Africa, and information on the predominant genotypes of Mycobacterium tuberculosis circulating in the country are important to better understand the epidemic. This study determined the predominant strain lineages that cause TB in Mozambique. Results A total of 445 M. tuberculosis isolates from seven different provinces of Mozambique were characterized by spoligotyping and resulting profiles were compared with the international spoligotyping database SITVIT2. The four most predominant lineages observed were: the Latin-American Mediterranean (LAM, n = 165 or 37%); the East African-Indian (EAI, n = 132 or 29.7%); an evolutionary recent but yet ill-defined T clade, (n = 52 or 11.6%); and the globally-emerging Beijing clone, (n = 31 or 7%). A high spoligotype diversity was found for the EAI, LAM and T lineages. Conclusions The TB epidemic in Mozambique is caused by a wide diversity of spoligotypes with predominance of LAM, EAI, T and Beijing lineages. PMID:20663126

  8. Genotype heterogeneity of Mycobacterium tuberculosis within geospatial hotspots suggests foci of imported infection in Sydney, Australia.

    PubMed

    Gurjav, Ulziijargal; Jelfs, Peter; Hill-Cawthorne, Grant A; Marais, Ben J; Sintchenko, Vitali

    2016-06-01

    In recent years the State of New South Wales (NSW), Australia, has maintained a low tuberculosis incidence rate with little evidence of local transmission. Nearly 90% of notified tuberculosis cases occurred in people born in tuberculosis-endemic countries. We analyzed geographic, epidemiological and genotypic data of all culture-confirmed tuberculosis cases to identify the bacterial and demographic determinants of tuberculosis hotspot areas in NSW. Standard 24-loci mycobacterium interspersed repetitive unit-variable number tandem repeat (MIRU-24) typing was performed on all isolates recovered between 2009 and 2013. In total 1692/1841 (91.9%) cases with confirmed Mycobacterium tuberculosis infection had complete MIRU-24 and demographic data and were included in the study. Despite some year-to-year variability, spatio-temporal analysis identified four tuberculosis hotspots. The incidence rate and the relative risk of tuberculosis in these hotspots were 2- to 10-fold and 4- to 8-fold higher than the state average, respectively. MIRU-24 profiles of M. tuberculosis isolates associated with these hotspots revealed high levels of heterogeneity. This suggests that these spatio-temporal hotspots, within this low incidence setting, can represent areas of predominantly imported infection rather than clusters of cases due to local transmission. These findings provide important epidemiological insight and demonstrate the value of combining tuberculosis genotyping and spatiotemporal data to guide better-targeted public health interventions. PMID:26187743

  9. Inhibitors Selective for Mycobacterial Versus Human Proteasomes

    SciTech Connect

    Lin, G.; Li, D; Sorio de Carvalho, L; Deng, H; Tao, H; Vogt, G; Wu, K; Schneider, J; Chidawanyika, T; et. al.

    2009-01-01

    Many anti-infectives inhibit the synthesis of bacterial proteins, but none selectively inhibits their degradation. Most anti-infectives kill replicating pathogens, but few preferentially kill pathogens that have been forced into a non-replicating state by conditions in the host. To explore these alternative approaches we sought selective inhibitors of the proteasome of Mycobacterium tuberculosis. Given that the proteasome structure is extensively conserved, it is not surprising that inhibitors of all chemical classes tested have blocked both eukaryotic and prokaryotic proteasomes, and no inhibitor has proved substantially more potent on proteasomes of pathogens than of their hosts. Here we show that certain oxathiazol-2-one compounds kill non-replicating M.?tuberculosis and act as selective suicide-substrate inhibitors of the M.?tuberculosis proteasome by cyclocarbonylating its active site threonine. Major conformational changes protect the inhibitor-enzyme intermediate from hydrolysis, allowing formation of an oxazolidin-2-one and preventing regeneration of active protease. Residues outside the active site whose hydrogen bonds stabilize the critical loop before and after it moves are extensively non-conserved. This may account for the ability of oxathiazol-2-one compounds to inhibit the mycobacterial proteasome potently and irreversibly while largely sparing the human homologue.

  10. TuberQ: a Mycobacterium tuberculosis protein druggability database

    PubMed Central

    Radusky, Leandro; Lanzarotti, Esteban; Luque, Javier; Barril, Xavier; Marti, Marcelo A.; Turjanski, Adrián G.

    2014-01-01

    In 2012 an estimated 8.6 million people developed tuberculosis (TB) and 1.3 million died from the disease [including 320 000 deaths among human immunodeficiency virus (HIV)-positive people]. There is an urgent need for new anti-TB drugs owing to the following: the fact that current treatments have severe side effects, the increasing emergence of multidrug-resistant strains of Mycobacterium tuberculosis (Mtb), the negative drug–drug interactions with certain HIV (or other disease) treatments and the ineffectiveness against dormant Mtb. In this context we present here the TuberQ database, a novel resource for all researchers working in the field of drug development in TB. The main feature of TuberQ is to provide a druggability analysis of Mtb proteins in a consistent and effective manner, contributing to a better selection of potential drug targets for screening campaigns and the analysis of targets for structure-based drug design projects. The structural druggability analysis is combined with features related to the characteristics of putative inhibitor binding pockets and with functional and biological data of proteins. The structural analysis is performed on all available unique Mtb structures and high-quality structural homology-based models. This information is shown in an interactive manner, depicting the protein structure, the pockets and the associated characteristics for each protein. TuberQ also provides information about gene essentiality information, as determined from whole cell–based knockout experiments, and expression information obtained from microarray experiments done in different stress-related conditions. We hope that TuberQ will be a powerful tool for researchers working in TB and eventually will lead to the identification of novel putative targets and progresses in therapeutic activities. Database URL: http://tuberq.proteinq.com.ar/ PMID:24816183

  11. Mycobacterium tuberculosis Zinc Metalloprotease-1 Assists Mycobacterial Dissemination in Zebrafish.

    PubMed

    Vemula, Mani H; Medisetti, Raghavender; Ganji, Rakesh; Jakkala, Kiran; Sankati, Swetha; Chatti, Kiranam; Banerjee, Sharmistha

    2016-01-01

    Zinc metalloprotease-1 (Zmp1) from Mycobacterium tuberculosis (M.tb), the tuberculosis (TB) causing bacillus, is a virulence factor involved in inflammasome inactivation and phagosome maturation arrest. We earlier reported that Zmp1 was secreted under granuloma-like stress conditions, induced Th2 cytokine microenvironment and was highly immunogenic in TB patients as evident from high anti-Zmp1 antibody titers in their sera. In this study, we deciphered a new physiological role of Zmp1 in mycobacterial dissemination. Exogenous treatment of THP-1 cells with 500 nM and 1 μM of recombinant Zmp1 (rZmp1) resulted in necrotic cell death. Apart from inducing secretion of necrotic cytokines, TNFα, IL-6, and IL-1β, it also induced the release of chemotactic chemokines, MCP-1, MIP-1β, and IL-8, suggesting its likely function in cell migration and mycobacterial dissemination. This was confirmed by Gap closure and Boyden chamber assays, where Zmp1 treated CHO or THP-1 cells showed ∼2 fold increased cell migration compared to the untreated cells. Additionally, Zebrafish-M. marinum based host-pathogen model was used to study mycobacterial dissemination in vivo. Td-Tomato labeled M. marinum (TdM. marinum) when injected with rZmp1 showed increased dissemination to tail region from the site of injection as compared to the untreated control fish in a dose-dependent manner. Summing up these observations along with the earlier reports, we propose that Zmp1, a multi-faceted protein, when released by mycobacteria in granuloma, may lead to necrotic cell damage and release of chemotactic chemokines by surrounding infected macrophages, attracting new immune cells, which in turn may lead to fresh cellular infections, thus assisting mycobacterial dissemination. PMID:27621726

  12. Mixed Infections and Rifampin Heteroresistance among Mycobacterium tuberculosis Clinical Isolates

    PubMed Central

    Zheng, Chao; Li, Song; Luo, Zhongyue; Pi, Rui; Sun, Honghu; He, Qingxia; Tang, Ke; Luo, Mei; Li, Yuqing; Couvin, David; Rastogi, Nalin

    2015-01-01

    Mixed infections and heteroresistance of Mycobacterium tuberculosis contribute to the difficulty of diagnosis, treatment, and control of tuberculosis. However, there is still no proper solution for these issues. This study aimed to investigate the potential relationship between mixed infections and heteroresistance and to determine the high-risk groups related to these factors. A total of 499 resistant and susceptible isolates were subjected to spoligotyping and 24-locus variable-number tandem repeat methods to analyze their genotypic lineages and the occurrence of mixed infections. Two hundred ninety-two randomly selected isolates were sequenced on their rpoB gene to examine mutations and heteroresistance. The results showed that 12 patients had mixed infections, and the corresponding isolates belonged to Manu2 (n = 8), Beijing (n = 2), T (n = 1), and unknown (n = 1) lineages. Manu2 was found to be significantly associated with mixed infections (odds ratio, 47.72; confidence interval, 9.68 to 235.23; P < 0.01). Four isolates (1.37%) were confirmed to be heteroresistant, which was caused by mixed infections in three (75%) isolates; these belonged to Manu2. Additionally, 3.8% of the rifampin-resistant isolates showing no mutation in the rpoB gene were significantly associated with mixed infections (χ2, 56.78; P < 0.01). This study revealed for the first time that Manu2 was the predominant group in the cases of mixed infections, and this might be the main reason for heteroresistance and a possible mechanism for isolates without any mutation in the rpoB gene to become rifampin resistant. Further studies should focus on this lineage to clarify its relevance to mixed infections. PMID:25903578

  13. Pyrazinamide resistance in Mycobacterium tuberculosis: Review and update.

    PubMed

    Njire, Moses; Tan, Yaoju; Mugweru, Julius; Wang, Changwei; Guo, Jintao; Yew, WingWai; Tan, Shouyong; Zhang, Tianyu

    2016-03-01

    The global control and management of tuberculosis (TB) is faced with the formidable challenge of worsening scenarios of drug-resistant disease. Pyrazinamide (PZA) is an indispensable first-line drug used for the treatment of TB. It plays a key role in reducing TB relapse rates, shortening the course of the disease treatment from 9-12 months to 6 months, and the treatment of patients infected with bacillary strains that are resistant to at least isoniazid and rifampicin. Additionally, it is the only first-line anti-TB drug most likely to be maintained in all new regimens, which are aimed at reducing the treatment period of susceptible, multi-drug resistant and extensively drug-resistant TB. It has a preferential sterilizing activity against non-replicating persister bacilli with low metabolism at acid pH in vitro or in vivo during active inflammation where other drugs may not act so well. PZA seem to have a non-specific cellular target and instead, exerts its anti-mycobacterial effect by disrupting the membrane energetics, the trans-translation process, acidification of the cytoplasm and perhaps coenzyme A synthesis, which is required for survival of Mycobacterium tuberculosis (MTB) persisters. Indeed, the emergence of MTB strains resistant to PZA represents an important clinical and public health problem. The essential role of PZA in TB treatment underlines the need for accurate and rapid detection of its resistance. This article presents an updated review of the molecular mechanisms of drug action and resistance in MTB against PZA, commenting on the several research gaps and proposed drug targets for PZA. PMID:26521205

  14. Mycobacterium tuberculosis Zinc Metalloprotease-1 Assists Mycobacterial Dissemination in Zebrafish

    PubMed Central

    Vemula, Mani H.; Medisetti, Raghavender; Ganji, Rakesh; Jakkala, Kiran; Sankati, Swetha; Chatti, Kiranam; Banerjee, Sharmistha

    2016-01-01

    Zinc metalloprotease-1 (Zmp1) from Mycobacterium tuberculosis (M.tb), the tuberculosis (TB) causing bacillus, is a virulence factor involved in inflammasome inactivation and phagosome maturation arrest. We earlier reported that Zmp1 was secreted under granuloma-like stress conditions, induced Th2 cytokine microenvironment and was highly immunogenic in TB patients as evident from high anti-Zmp1 antibody titers in their sera. In this study, we deciphered a new physiological role of Zmp1 in mycobacterial dissemination. Exogenous treatment of THP-1 cells with 500 nM and 1 μM of recombinant Zmp1 (rZmp1) resulted in necrotic cell death. Apart from inducing secretion of necrotic cytokines, TNFα, IL-6, and IL-1β, it also induced the release of chemotactic chemokines, MCP-1, MIP-1β, and IL-8, suggesting its likely function in cell migration and mycobacterial dissemination. This was confirmed by Gap closure and Boyden chamber assays, where Zmp1 treated CHO or THP-1 cells showed ∼2 fold increased cell migration compared to the untreated cells. Additionally, Zebrafish-M. marinum based host–pathogen model was used to study mycobacterial dissemination in vivo. Td-Tomato labeled M. marinum (TdM. marinum) when injected with rZmp1 showed increased dissemination to tail region from the site of injection as compared to the untreated control fish in a dose-dependent manner. Summing up these observations along with the earlier reports, we propose that Zmp1, a multi-faceted protein, when released by mycobacteria in granuloma, may lead to necrotic cell damage and release of chemotactic chemokines by surrounding infected macrophages, attracting new immune cells, which in turn may lead to fresh cellular infections, thus assisting mycobacterial dissemination. PMID:27621726

  15. Whole genome sequencing of Mycobacterium tuberculosis SB24 isolated from Sabah, Malaysia.

    PubMed

    Philip, Noraini; Rodrigues, Kenneth Francis; William, Timothy; John, Daisy Vanitha

    2016-09-01

    Mycobacterium tuberculosis (M. tuberculosis) is the causative agent of tuberculosis (TB) that causes millions of death every year. We have sequenced the genome of M. tuberculosis isolated from cerebrospinal fluid (CSF) of a patient diagnosed with tuberculous meningitis (TBM). The isolated strain was referred as M. tuberculosis SB24. Genomic DNA of the M. tuberculosis SB24 was extracted and subjected to whole genome sequencing using PacBio platform. The draft genome size of M. tuberculosis SB24 was determined to be 4,452,489 bp with a G + C content of 65.6%. The whole genome shotgun project has been deposited in NCBI SRA under the accession number SRP076503. PMID:27556011

  16. Differential influence of nutrient-starved Mycobacterium tuberculosis on adaptive immunity results in progressive tuberculosis disease and pathology.

    PubMed

    Dietrich, Jes; Roy, Sugata; Rosenkrands, Ida; Lindenstrøm, Thomas; Filskov, Jonathan; Rasmussen, Erik Michael; Cassidy, Joseph; Andersen, Peter

    2015-12-01

    When infected with Mycobacterium tuberculosis, most individuals will remain clinically healthy but latently infected. Latent infection has been proposed to partially involve M. tuberculosis in a nonreplicating stage, which therefore represents an M. tuberculosis phenotype that the immune system most likely will encounter during latency. It is therefore relevant to examine how this particular nonreplicating form of M. tuberculosis interacts with the host immune system. To study this, we first induced a state of nonreplication through prolonged nutrient starvation of M. tuberculosis in vitro. This resulted in nonreplicating persistence even after prolonged culture in phosphate-buffered saline. Infection with either exponentially growing M. tuberculosis or nutrient-starved M. tuberculosis resulted in similar lung CFU levels in the first phase of the infection. However, between week 3 and 6 postinfection, there was a very pronounced increase in bacterial levels and associated lung pathology in nutrient-starved-M. tuberculosis-infected mice. This was associated with a shift from CD4 T cells that coexpressed gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) or IFN-γ, TNF-α, and interleukin-2 to T cells that only expressed IFN-γ. Thus, nonreplicating M. tuberculosis induced through nutrient starvation promotes a bacterial form that is genetically identical to exponentially growing M. tuberculosis yet characterized by a differential impact on the immune system that may be involved in undermining host antimycobacterial immunity and facilitate increased pathology and transmission. PMID:26416911

  17. Mycobacterium tuberculosis infection as a zoonotic disease: transmission between humans and elephants.

    PubMed Central

    Michalak, K.; Austin, C.; Diesel, S.; Bacon, M. J.; Zimmerman, P.; Maslow, J. N.

    1998-01-01

    Between 1994 and 1996, three elephants from an exotic animal farm in Illinois died of pulmonary disease due to Mycobacterium tuberculosis. In October 1996, a fourth living elephant was culture-positive for M. tuberculosis. Twenty-two handlers at the farm were screened for tuberculosis (TB); eleven had positive reactions to intradermal injection with purified protein derivative. One had smear-negative, culture-positive active TB. DNA fingerprint comparison by IS6110 and TBN12 typing showed that the isolates from the four elephants and the handler with active TB were the same strain. This investigation indicates transmission of M. tuberculosis between humans and elephants. PMID:9621200

  18. Differentiation of slowly growing Mycobacterium species, including Mycobacterium tuberculosis, by gene amplification and restriction fragment length polymorphism analysis.

    PubMed Central

    Plikaytis, B B; Plikaytis, B D; Yakrus, M A; Butler, W R; Woodley, C L; Silcox, V A; Shinnick, T M

    1992-01-01

    A two-step assay combining a gene amplification step and a restriction fragment length polymorphism analysis was developed to differentiate the Mycobacterium species that account for greater than 90% of potentially pathogenic isolates and greater than 86% of all isolates in clinical laboratories in the United States. These species are M. tuberculosis, M. bovis, M. avium, M. intracellulare, M. kansasii, and M. gordonae. With lysates of pure cultures as the template, two oligonucleotide primers that amplified an approximately 1,380-bp portion of the hsp65 gene from all 139 strains of 19 Mycobacterium species tested, but not from the 19 non-Mycobacterium species tested, were identified. Digestion of the amplicons from 126 strains of the six most commonly isolated Mycobacterium species with the restriction enzymes BstNI and XhoI in separate reactions generated restriction fragment patterns that were distinctive for each of these species, except for those of M. tuberculosis and M. bovis, which were not distinguishable. By including size standards in each sample, the restriction fragment profiles could be normalized to a fixed distance and the similarities of patterns could be calculated by using a computer-aided comparison program. The availability of this data base should enable the identification of an unknown Mycobacterium strain to the species level by a comparison of the restriction fragment pattern of the unknown with the data base of known patterns. Images PMID:1352786

  19. Mycobacterium aurum is Unable to Survive Mycobacterium tuberculosis Latency Associated Stress Conditions: Implications as Non-suitable Model Organism.

    PubMed

    Sood, Shivani; Yadav, Anant; Shrivastava, Rahul

    2016-06-01

    Mycobacterium tuberculosis manages to remain latent in the human body regardless of extensive chemotherapy. Complete eradication of tuberculosis (TB) requires treatment strategies targeted against latent form of infection, in addition to the current regimen of antimycobacterials. Many in vitro and in vivo models have been proposed to imitate latent TB infection, yet none of them is able to completely mimic latent infection state of M. tuberculosis. Highly infectious nature of the pathogen requiring BSL3 facilities and its long generation time further add to complications. M. aurum has been proposed as an important model organism for high throughput screening of drugs and exhibits high genomic similarity with that of M. tuberculosis. Thus, the present study was undertaken to explore if M. aurum could be used as a surrogate organism for studies related to M. tuberculosis latent infection. M. aurum was subjected to in vitro conditions of oxygen depletion, lack of nutrients and acidic stress encountered by latent M. tuberculosis bacteria. CFU count of M. aurum cells along with any change in cell shape and size was recorded at regular intervals during the stress conditions. M. aurum cells were unable to survive for extended periods under all three conditions used in the study. Thus, our studies suggest that M. aurum is not a suitable organism to mimic M. tuberculosis persistent infection under in vitro conditions, and further studies are required on different species for the establishment of a fast growing species as a suitable model for M. tuberculosis persistent infection. PMID:27570312

  20. Rapid detection of Mycobacterium tuberculosis by recombinase polymerase amplification.

    PubMed

    Boyle, David S; McNerney, Ruth; Teng Low, Hwee; Leader, Brandon Troy; Pérez-Osorio, Ailyn C; Meyer, Jessica C; O'Sullivan, Denise M; Brooks, David G; Piepenburg, Olaf; Forrest, Matthew S

    2014-01-01

    Improved access to effective tests for diagnosing tuberculosis (TB) has been designated a public health priority by the World Health Organisation. In high burden TB countries nucleic acid based TB tests have been restricted to centralised laboratories and specialised research settings. Requirements such as a constant electrical supply, air conditioning and skilled, computer literate operators prevent implementation of such tests in many settings. Isothermal DNA amplification technologies permit the use of simpler, less energy intensive detection platforms more suited to low resource settings that allow the accurate diagnosis of a disease within a short timeframe. Recombinase Polymerase Amplification (RPA) is a rapid, low temperature isothermal DNA amplification reaction. We report here RPA-based detection of Mycobacterium tuberculosis complex (MTC) DNA in <20 minutes at 39 °C. Assays for two MTC specific targets were investigated, IS6110 and IS1081. When testing purified MTC genomic DNA, limits of detection of 6.25 fg (IS6110) and 20 fg (IS1081)were consistently achieved. When testing a convenience sample of pulmonary specimens from suspected TB patients, RPA demonstrated superior accuracy to indirect fluorescence microscopy. Compared to culture, sensitivities for the IS1081 RPA and microscopy were 91.4% (95%CI: 85, 97.9) and 86.1% (95%CI: 78.1, 94.1) respectively (n = 71). Specificities were 100% and 88.6% (95% CI: 80.8, 96.1) respectively. For the IS6110 RPA and microscopy sensitivities of 87.5% (95%CI: 81.7, 93.2) and 70.8% (95%CI: 62.9, 78.7) were obtained (n = 90). Specificities were 95.4 (95% CI: 92.3,98.1) and 88% (95% CI: 83.6, 92.4) respectively. The superior specificity of RPA for detecting tuberculosis was due to the reduced ability of fluorescence microscopy to distinguish Mtb complex from other acid fast bacteria. The rapid nature of the RPA assay and its low energy requirement compared to other amplification technologies suggest RPA-based TB assays

  1. Structure and function of a 40,000-molecular-weight protein antigen of Mycobacterium tuberculosis.

    PubMed Central

    Andersen, A B; Andersen, P; Ljungqvist, L

    1992-01-01

    A gene encoding a protein antigen from Mycobacterium tuberculosis with a molecular weight of 40,000 has been sequenced. On the basis of sequence homology and functional analyses, we demonstrated that the protein is an L-alanine dehydrogenase (EC 1.4.1.1). The enzyme was demonstrated in M. tuberculosis and Mycobacterium marinum but not in Mycobacterium bovis BCG. The enzyme may play a role in cell wall synthesis because L-alanine is an important constituent of the peptidoglycan layer. Although no consensus signal sequence was identified, we found evidence which suggests that the enzyme is secreted across the cell membrane. The enzyme was characterized and purified by chromatography, thus enabling further studies of its role in virulence and interaction with the immune system of M. tuberculosis-infected individuals. Images PMID:1587598

  2. Nucleotide triphosphate promiscuity in Mycobacterium tuberculosis dethiobiotin synthetase.

    PubMed

    Salaemae, Wanisa; Yap, Min Y; Wegener, Kate L; Booker, Grant W; Wilce, Matthew C J; Polyak, Steven W

    2015-05-01

    Dethiobiotin synthetase (DTBS) plays a crucial role in biotin biosynthesis in microorganisms, fungi, and plants. Due to its importance in bacterial pathogenesis, and the absence of a human homologue, DTBS is a promising target for the development of new antibacterials desperately needed to combat antibiotic resistance. Here we report the first X-ray structure of DTBS from Mycobacterium tuberculosis (MtDTBS) bound to a nucleotide triphosphate (CTP). The nucleoside base is stabilized in its pocket through hydrogen-bonding interactions with the protein backbone, rather than amino acid side chains. This resulted in the unexpected finding that MtDTBS could utilise ATP, CTP, GTP, ITP, TTP, or UTP with similar Km and kcat values, although the enzyme had the highest affinity for CTP in competitive binding and surface plasmon resonance assays. This is in contrast to other DTBS homologues that preferentially bind ATP primarily through hydrogen-bonds between the purine base and the carboxamide side chain of a key asparagine. Mutational analysis performed alongside in silico experiments revealed a gate-keeper role for Asn175 in Escherichia coli DTBS that excludes binding of other nucleotide triphosphates. Here we provide evidence to show that MtDTBS has a broad nucleotide specificity due to the absence of the gate-keeper residue. PMID:25801336

  3. Transcriptional responses of Mycobacterium tuberculosis to lung surfactant

    PubMed Central

    Schwab, Ute; Rohde, Kyle H.; Wang, Zhengdong; Chess, Patricia R.; Notter, Robert H.; Russell, David G.

    2009-01-01

    This study uses microarray analyses to examine gene expression profiles for Mycobacterium tuberculosis (Mtb) induced by exposure in vitro to bovine lung surfactant preparations that vary in apoprotein content: (i) whole lung surfactant (WLS) containing the complete mix of endogenous lipids and surfactant proteins (SP)-A, -B, -C, and -D; (ii) extracted lung surfactant (CLSE) containing lipids plus SP-B and -C; (iii) column-purified surfactant lipids (PPL) containing no apoproteins, and (iv) purified human SP-A. Exposure to WLS evoked a multitude of transcriptional responses in Mtb, with 52 genes up-regulated and 23 genes down-regulated at 30 min exposure, plus 146 genes up-regulated and 27 genes down-regulated at 2 h. Notably, WLS rapidly induced several membrane-associated lipases that presumptively act on surfactant lipids as substrates, and a large number of genes involved in the synthesis of phthiocerol dimycocerosate (PDIM), a cell wall component known to be important in macrophage interactions and Mtb virulence. Exposure of Mtb to CLSE, PPL, or purified SP-A caused a substantially weaker transcriptional response (≤20 genes were induced) suggesting that interactions among multiple lipid-protein components of WLS may contribute to its effects on Mtb transcription. PMID:19272305

  4. Identification of widespread adenosine nucleotide binding in Mycobacterium tuberculosis

    SciTech Connect

    Ansong, Charles; Ortega, Corrie; Payne, Samuel H.; Haft, Daniel H.; Chauvigne-Hines, Lacie M.; Lewis, Michael P.; Ollodart, Anja R.; Purvine, Samuel O.; Shukla, Anil K.; Fortuin, Suereta; Smith, Richard D.; Adkins, Joshua N.; Grundner, Christoph; Wright, Aaron T.

    2013-01-24

    The annotation of protein function is almost completely performed by in silico approaches. However, computational prediction of protein function is frequently incomplete and error prone. In Mycobacterium tuberculosis (Mtb), ~25% of all genes have no predicted function and are annotated as hypothetical proteins. This lack of functional information severely limits our understanding of Mtb pathogenicity. Current tools for experimental functional annotation are limited and often do not scale to entire protein families. Here, we report a generally applicable chemical biology platform to functionally annotate bacterial proteins by combining activity-based protein profiling (ABPP) and quantitative LC-MS-based proteomics. As an example of this approach for high-throughput protein functional validation and discovery, we experimentally annotate the families of ATP-binding proteins in Mtb. Our data experimentally validate prior in silico predictions of >250 ATPases and adenosine nucleotide-binding proteins, and reveal 73 hypothetical proteins as novel ATP-binding proteins. We identify adenosine cofactor interactions with many hypothetical proteins containing a diversity of unrelated sequences, providing a new and expanded view of adenosine nucleotide binding in Mtb. Furthermore, many of these hypothetical proteins are both unique to Mycobacteria and essential for infection, suggesting specialized functions in mycobacterial physiology and pathogenicity. Thus, we provide a generally applicable approach for high throughput protein function discovery and validation, and highlight several ways in which application of activity-based proteomics data can improve the quality of functional annotations to facilitate novel biological insights.

  5. Role of Cathepsins in Mycobacterium tuberculosis Survival in Human Macrophages.

    PubMed

    Pires, David; Marques, Joana; Pombo, João Palma; Carmo, Nuno; Bettencourt, Paulo; Neyrolles, Olivier; Lugo-Villarino, Geanncarlo; Anes, Elsa

    2016-01-01

    Cathepsins are proteolytic enzymes that function in the endocytic pathway, especially in lysosomes, where they contribute directly to pathogen killing or indirectly, by their involvement in the antigen presentation pathways. Mycobacterium tuberculosis (MTB) is a facultative intracellular pathogen that survives inside the macrophage phagosomes by inhibiting their maturation to phagolysosomes and thus avoiding a low pH and protease-rich environment. We previously showed that mycobacterial inhibition of the proinflammatory transcription factor NF-κB results in impaired delivery of lysosomal enzymes to phagosomes and reduced pathogen killing. Here, we elucidate how MTB also controls cathepsins and their inhibitors, cystatins, at the level of gene expression and proteolytic activity. MTB induced a general down-regulation of cathepsin expression in infected cells, and inhibited IFNγ-mediated increase of cathepsin mRNA. We further show that a decrease in cathepsins B, S and L favours bacterial survival within human primary macrophages. A siRNA knockdown screen of a large set of cathepsins revealed that almost half of these enzymes have a role in pathogen killing, while only cathepsin F coincided with MTB resilience. Overall, we show that cathepsins are important for the control of MTB infection, and as a response, it manipulates their expression and activity to favour its intracellular survival. PMID:27572605

  6. Tuberculosis (Mycobacterium microti) in wild field vole populations

    PubMed Central

    BURTHE, S.; BENNETT, M.; KIPAR, A.; LAMBIN, X.; SMITH, A.; TELFER, S.; BEGON, M.

    2010-01-01

    SUMMARY Vole tuberculosis (TB; Mycobacterium microti) is an understudied endemic infection. Despite progressing slowly, it causes severe clinical pathology and overt symptoms in its rodent host. TB was monitored for 2 years in wild field voles in Kielder Forest, UK. The prevalence of characteristic cutaneous TB lesions was monitored longitudinally at 4 sites, with individuals live-trapped and repeatedly monitored. A prevalence of 5·2% of individuals with lesions was recorded (n=2791). In a cross-sectional study, 27 sites were monitored bi-annually, with TB assessed by post-mortem examination for macroscopic lesions, and by culture and histopathology. Seventy-nine voles (10·78%; n=733) were positive for mycobacteria, with the highest prevalence in spring (13·15%; n=327). TB prevalence varied, with between 0% and 50% of voles infected per site. Prevalence increased with age (mass), and apparent seasonality was due to a higher proportion of older animals in spring. Survival analysis supported this result, with cutaneous lesions only manifesting in the advanced stages of infection, and therefore only being found on older voles. The body condition of individuals with lesions declined at the time when the lesion was first recorded, when compared to individuals without lesions, suggesting there may be an acute phase of infection during its advanced stage. Although predicted survival following the appearance of a cutaneous lesion was lower than for uninfected individuals, this was not significant. PMID:18005472

  7. Tuberculosis (Mycobacterium microti) in wild field vole populations.

    PubMed

    Burthe, S; Bennett, M; Kipar, A; Lambin, X; Smith, A; Telfer, S; Begon, M

    2008-03-01

    Vole tuberculosis (TB; Mycobacterium microti) is an understudied endemic infection. Despite progressing slowly, it causes severe clinical pathology and overt symptoms in its rodent host. TB was monitored for 2 years in wild field voles in Kielder Forest, UK. The prevalence of characteristic cutaneous TB lesions was monitored longitudinally at 4 sites, with individuals live-trapped and repeatedly monitored. A prevalence of 5.2% of individuals with lesions was recorded (n=2791). In a cross-sectional study, 27 sites were monitored bi-annually, with TB assessed by post-mortem examination for macroscopic lesions, and by culture and histopathology. Seventy-nine voles (10.78%; n=733) were positive for mycobacteria, with the highest prevalence in spring (13.15%; n=327). TB prevalence varied, with between 0% and 50% of voles infected per site. Prevalence increased with age (mass), and apparent seasonality was due to a higher proportion of older animals in spring. Survival analysis supported this result, with cutaneous lesions only manifesting in the advanced stages of infection, and therefore only being found on older voles. The body condition of individuals with lesions declined at the time when the lesion was first recorded, when compared to individuals without lesions, suggesting there may be an acute phase of infection during its advanced stage. Although predicted survival following the appearance of a cutaneous lesion was lower than for uninfected individuals, this was not significant. PMID:18005472

  8. Genetic features of Mycobacterium tuberculosis modern Beijing sublineage

    PubMed Central

    Liu, Qingyun; Luo, Tao; Dong, Xinran; Sun, Gang; Liu, Zhu; Gan, Mingyun; Wu, Jie; Shen, Xin; Gao, Qian

    2016-01-01

    Mycobacterium tuberculosis (MTB) Beijing strains have caused a great concern because of their rapid emergence and increasing prevalence in worldwide regions. Great efforts have been made to investigate the pathogenic characteristics of Beijing strains such as hypervirulence, drug resistance and favoring transmission. Phylogenetically, MTB Beijing family was divided into modern and ancient sublineages. Modern Beijing strains displayed enhanced virulence and higher prevalence when compared with ancient Beijing strains, but the genetic basis for this difference remains unclear. In this study, by analyzing previously published sequencing data of 1082 MTB Beijing isolates, we determined the genetic changes that were commonly present in modern Beijing strains but absent in ancient Beijing strains. These changes include 44 single-nucleotide polymorphisms (SNPs) and two short genomic deletions. Through bioinformatics analysis, we demonstrated that these genetic changes had high probability of functional effects. For example, 4 genes were frameshifted due to premature stop mutation or genomic deletions, 19 nonsynonymous SNPs located in conservative codons, and there is a significant enrichment in regulatory network for all nonsynonymous mutations. Besides, three SNPs located in promoter regions were verified to alter downstream gene expressions. Our study precisely defined the genetic features of modern Beijing strains and provided interesting clues for future researches to elucidate the mechanisms that underlie this sublineage's successful expansion. These findings from the analysis of the modern Beijing sublineage could provide us a model to understand the dynamics of pathogenicity of MTB. PMID:26905026

  9. Mycobacterium tuberculosis Pyrazinamide Resistance Determinants: a Multicenter Study

    PubMed Central

    Cabibbe, Andrea M.; Feuerriegel, Silke; Casali, Nicola; Drobniewski, Francis; Rodionova, Yulia; Bakonyte, Daiva; Stakenas, Petras; Pimkina, Edita; Augustynowicz-Kopeć, Ewa; Degano, Massimo; Ambrosi, Alessandro; Hoffner, Sven; Mansjö, Mikael; Werngren, Jim; Rüsch-Gerdes, Sabine; Niemann, Stefan; Cirillo, Daniela M.

    2014-01-01

    ABSTRACT Pyrazinamide (PZA) is a prodrug that is converted to pyrazinoic acid by the enzyme pyrazinamidase, encoded by the pncA gene in Mycobacterium tuberculosis. Molecular identification of mutations in pncA offers the potential for rapid detection of pyrazinamide resistance (PZAr). However, the genetic variants are highly variable and scattered over the full length of pncA, complicating the development of a molecular test. We performed a large multicenter study assessing pncA sequence variations in 1,950 clinical isolates, including 1,142 multidrug-resistant (MDR) strains and 483 fully susceptible strains. The results of pncA sequencing were correlated with phenotype, enzymatic activity, and structural and phylogenetic data. We identified 280 genetic variants which were divided into four classes: (i) very high confidence resistance mutations that were found only in PZAr strains (85%), (ii) high-confidence resistance mutations found in more than 70% of PZAr strains, (iii) mutations with an unclear role found in less than 70% of PZAr strains, and (iv) mutations not associated with phenotypic resistance (10%). Any future molecular diagnostic assay should be able to target and identify at least the very high and high-confidence genetic variant markers of PZAr; the diagnostic accuracy of such an assay would be in the range of 89.5 to 98.8%. PMID:25336456

  10. Structural basis for benzothiazinone-mediated killing of Mycobacterium tuberculosis.

    PubMed

    Neres, João; Pojer, Florence; Molteni, Elisabetta; Chiarelli, Laurent R; Dhar, Neeraj; Boy-Röttger, Stefanie; Buroni, Silvia; Fullam, Elizabeth; Degiacomi, Giulia; Lucarelli, Anna Paola; Read, Randy J; Zanoni, Giuseppe; Edmondson, Dale E; De Rossi, Edda; Pasca, Maria Rosalia; McKinney, John D; Dyson, Paul J; Riccardi, Giovanna; Mattevi, Andrea; Cole, Stewart T; Binda, Claudia

    2012-09-01

    The benzothiazinone BTZ043 is a tuberculosis drug candidate with nanomolar whole-cell activity. BTZ043 targets the DprE1 catalytic component of the essential enzyme decaprenylphosphoryl-β-D-ribofuranose-2'-epimerase, thus blocking biosynthesis of arabinans, vital components of mycobacterial cell walls. Crystal structures of DprE1, in its native form and in a complex with BTZ043, reveal formation of a semimercaptal adduct between the drug and an active-site cysteine, as well as contacts to a neighboring catalytic lysine residue. Kinetic studies confirm that BTZ043 is a mechanism-based, covalent inhibitor. This explains the exquisite potency of BTZ043, which, when fluorescently labeled, localizes DprE1 at the poles of growing bacteria. Menaquinone can reoxidize the flavin adenine dinucleotide cofactor in DprE1 and may be the natural electron acceptor for this reaction in the mycobacterium. Our structural and kinetic analysis provides both insight into a critical epimerization reaction and a platform for structure-based design of improved inhibitors. PMID:22956199

  11. Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

    PubMed Central

    Linger, Yvonne; Kukhtin, Alexander; Golova, Julia; Perov, Alexander; Qu, Peter; Knickerbocker, Christopher; Cooney, Christopher G.; Chandler, Darrell P.

    2014-01-01

    Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice. PMID:24796567

  12. Pathogen ‘Roid Rage: Cholesterol Utilization by Mycobacterium tuberculosis

    PubMed Central

    Wipperman, Matthew F.; Sampson, Nicole S.; Thomas, Suzanne, T.

    2014-01-01

    The ability of science and medicine to control the pathogen Mycobacterium tuberculosis (Mtb) requires an understanding of the complex host environment within which it resides. Pathological and biological evidence overwhelmingly demonstrate how the mammalian steroid cholesterol is present throughout the course of infection. Better understanding Mtb requires a more complete understanding of how it utilizes molecules like cholesterol in this environment to sustain the infection of the host. Cholesterol uptake, catabolism, and broader utilization are important for maintenance of the pathogen in the host and it has been experimentally validated to contribute to virulence and pathogenesis. Cholesterol is catabolized by at least three distinct sub-pathways, two for the ring system and one for the side chain, yielding dozens of steroid intermediates with varying biochemical properties. Our ability to control this worldwide infectious agent requires a greater knowledge of how Mtb uses cholesterol to its advantage throughout the course of infection. Herein, the current state of knowledge of cholesterol metabolism by Mtb is reviewed from a biochemical perspective with a focus on the metabolic genes and pathways responsible for cholesterol steroid catabolism. PMID:24611808

  13. Role of Cathepsins in Mycobacterium tuberculosis Survival in Human Macrophages

    PubMed Central

    Pires, David; Marques, Joana; Pombo, João Palma; Carmo, Nuno; Bettencourt, Paulo; Neyrolles, Olivier; Lugo-Villarino, Geanncarlo; Anes, Elsa

    2016-01-01

    Cathepsins are proteolytic enzymes that function in the endocytic pathway, especially in lysosomes, where they contribute directly to pathogen killing or indirectly, by their involvement in the antigen presentation pathways. Mycobacterium tuberculosis (MTB) is a facultative intracellular pathogen that survives inside the macrophage phagosomes by inhibiting their maturation to phagolysosomes and thus avoiding a low pH and protease-rich environment. We previously showed that mycobacterial inhibition of the proinflammatory transcription factor NF-κB results in impaired delivery of lysosomal enzymes to phagosomes and reduced pathogen killing. Here, we elucidate how MTB also controls cathepsins and their inhibitors, cystatins, at the level of gene expression and proteolytic activity. MTB induced a general down-regulation of cathepsin expression in infected cells, and inhibited IFNγ-mediated increase of cathepsin mRNA. We further show that a decrease in cathepsins B, S and L favours bacterial survival within human primary macrophages. A siRNA knockdown screen of a large set of cathepsins revealed that almost half of these enzymes have a role in pathogen killing, while only cathepsin F coincided with MTB resilience. Overall, we show that cathepsins are important for the control of MTB infection, and as a response, it manipulates their expression and activity to favour its intracellular survival. PMID:27572605

  14. Phylogenetic analysis of vitamin B12-related metabolism in Mycobacterium tuberculosis

    PubMed Central

    Young, Douglas B.; Comas, Iñaki; de Carvalho, Luiz P. S.

    2015-01-01

    Comparison of genome sequences from clinical isolates of Mycobacterium tuberculosis with phylogenetically-related pathogens Mycobacterium marinum, Mycobacterium kansasii, and Mycobacterium leprae reveals diversity amongst genes associated with vitamin B12-related metabolism. Diversity is generated by gene deletion events, differential acquisition of genes by horizontal transfer, and single nucleotide polymorphisms (SNPs) with predicted impact on protein function and transcriptional regulation. Differences in the B12 synthesis pathway, methionine biosynthesis, fatty acid catabolism, and DNA repair and replication are consistent with adaptations to different environmental niches and pathogenic lifestyles. While there is no evidence of further gene acquisition during expansion of the M. tuberculosis complex, the emergence of other forms of genetic diversity provides insights into continuing host-pathogen co-evolution and has the potential to identify novel targets for disease intervention. PMID:25988174

  15. Necrosis of lung epithelial cells during infection with Mycobacterium tuberculosis is preceded by cell permeation.

    PubMed

    Dobos, K M; Spotts, E A; Quinn, F D; King, C H

    2000-11-01

    Mycobacterium tuberculosis establishes infection, progresses towards disease, and is transmitted from the alveolus of the lung. However, the role of the alveolar epithelium in any of these pathogenic processes of tuberculosis is unclear. In this study, lung epithelial cells (A549) were used as a model in which to examine cytotoxicity during infection with either virulent or avirulent mycobacteria in order to further establish the role of the lung epithelium during tuberculosis. Infection of A549 cells with M. tuberculosis strains Erdman and CDC1551 demonstrated significant cell monolayer clearing, whereas infection with either Mycobacterium bovis BCG or Mycobacterium smegmatis LR222 did not. Clearing of M. tuberculosis-infected A549 cells correlated to necrosis, not apoptosis. Treatment of M. tuberculosis-infected A549 cells with streptomycin, but not cycloheximide, demonstrated a significant reduction in the necrosis of A549 cell monolayers. This mycobacterium-induced A549 necrosis did not correlate to higher levels of intracellular or extracellular growth by the mycobacteria during infection. Staining of infected cells with propidium iodide demonstrated that M. tuberculosis induced increased permeation of A549 cell membranes within 24 h postinfection. Quantitation of lactate dehydrogenase (LDH) release from infected cells further demonstrated that cell permeation was specific to M. tuberculosis infection and correlated to A549 cellular necrosis. Inactivated M. tuberculosis or its subcellular fractions did not result in A549 necrosis or LDH release. These studies demonstrate that lung epithelial cell cytotoxicity is specific to infection by virulent mycobacteria and is caused by cellular necrosis. This necrosis is not a direct correlate of mycobacterial growth or of the expression of host cell factors, but is preceded by permeation of the A549 cell membrane and requires infection with live bacilli. PMID:11035739

  16. Sputum is a surrogate for bronchoalveolar lavage for monitoring Mycobacterium tuberculosis transcriptional profiles in TB patients.

    PubMed

    Garcia, Benjamin J; Loxton, Andre G; Dolganov, Gregory M; Van, Tran T; Davis, J Lucian; de Jong, Bouke C; Voskuil, Martin I; Leach, Sonia M; Schoolnik, Gary K; Walzl, Gerhard; Strong, Michael; Walter, Nicholas D

    2016-09-01

    Pathogen-targeted transcriptional profiling in human sputum may elucidate the physiologic state of Mycobacterium tuberculosis (M. tuberculosis) during infection and treatment. However, whether M. tuberculosis transcription in sputum recapitulates transcription in the lung is uncertain. We therefore compared M. tuberculosis transcription in human sputum and bronchoalveolar lavage (BAL) samples from 11 HIV-negative South African patients with pulmonary tuberculosis. We additionally compared these clinical samples with in vitro log phase aerobic growth and hypoxic non-replicating persistence (NRP-2). Of 2179 M. tuberculosis transcripts assayed in sputum and BAL via multiplex RT-PCR, 194 (8.9%) had a p-value <0.05, but none were significant after correction for multiple testing. Categorical enrichment analysis indicated that expression of the hypoxia-responsive DosR regulon was higher in BAL than in sputum. M. tuberculosis transcription in BAL and sputum was distinct from both aerobic growth and NRP-2, with a range of 396-1020 transcripts significantly differentially expressed after multiple testing correction. Collectively, our results indicate that M. tuberculosis transcription in sputum approximates M. tuberculosis transcription in the lung. Minor differences between M. tuberculosis transcription in BAL and sputum suggested lower oxygen concentrations or higher nitric oxide concentrations in BAL. M. tuberculosis-targeted transcriptional profiling of sputa may be a powerful tool for understanding M. tuberculosis pathogenesis and monitoring treatment responses in vivo. PMID:27553415

  17. High-throughput Method of One-Step DNA Isolation for PCR Diagnostics of Mycobacterium tuberculosis.

    PubMed

    Kapustin, D V; Prostyakova, A I; Alexeev, Ya I; Varlamov, D A; Zubov, V P; Zavriev, S K

    2014-04-01

    The efficiency of one-step and multi-step protocols of DNA isolation from lysed sputum samples containing the Mycobacterium tuberculosis complex has been compared. DNA was isolated using spin-cartridges containing a special silica-based sorbent modified with fluoroplast and polyaniline, or using an automated isolation system. One-step isolation using the obtained sorbent has been shown to ensure a significantly lower DNA loss and higher sensitivity in the PCR detection of Mycobacterium tuberculosis as compared to a system based on sorption and desorption of nucleic acids during the isolation. PMID:25093111

  18. Comparative Genomics and Proteomic Analysis of Four Non-tuberculous Mycobacterium Species and Mycobacterium tuberculosis Complex: Occurrence of Shared Immunogenic Proteins

    PubMed Central

    Gcebe, Nomakorinte; Michel, Anita; Gey van Pittius, Nicolaas C.; Rutten, Victor

    2016-01-01

    The Esx and PE/PPE families of proteins are among the most immunodominant mycobacterial antigens and have thus been the focus of research to develop vaccines and immunological tests for diagnosis of bovine and human tuberculosis, mainly caused by Mycobacterium bovis and Mycobacterium tuberculosis, respectively. In non-tuberculous mycobacteria (NTM), multiple copies of genes encoding homologous proteins have mainly been identified in pathogenic Mycobacterium species phylogenically related to Mycobacterium tuberculosis and Mycobacterium bovis. Only ancestral copies of these genes have been identified in nonpathogenic NTM species like Mycobacterium smegmatis, Mycobacterium sp. KMS, Mycobacterium sp. MCS, and Mycobacterium sp. JLS. In this study we elucidated the genomes of four nonpathogenic NTM species, viz Mycobacterium komanii sp. nov., Mycobacterium malmesburii sp. nov., Mycobacterium nonchromogenicum, and Mycobacterium fortuitum ATCC 6841. These genomes were investigated for genes encoding for the Esx and PE/PPE (situated in the esx cluster) family of proteins as well as adjacent genes situated in the ESX-1 to ESX-5 regions. To identify proteins actually expressed, comparative proteomic analyses of purified protein derivatives from three of the NTM as well as Mycobacterium kansasii ATCC 12478 and the commercially available purified protein derivatives from Mycobacterium bovis and Mycobacterium avium was performed. The genomic analysis revealed the occurrence in each of the four NTM, orthologs of the genes encoding for the Esx family, the PE and PPE family proteins in M. bovis and M. tuberculosis. The identification of genes of the ESX-1, ESX-3, and ESX-4 region including esxA, esxB, ppe68, pe5, and pe35 adds to earlier reports of these genes in nonpathogenic NTM like M. smegmatis, Mycobacterium sp. JLS and Mycobacterium KMS. This report is also the first to identify esxN gene situated within the ESX-5 locus in M. nonchromogenicum. Our proteomics analysis

  19. An integrated map of the genome of the tubercle bacillus, Mycobacterium tuberculosis H37Rv, and comparison with Mycobacterium leprae.

    PubMed Central

    Philipp, W J; Poulet, S; Eiglmeier, K; Pascopella, L; Balasubramanian, V; Heym, B; Bergh, S; Bloom, B R; Jacobs, W R; Cole, S T

    1996-01-01

    An integrated map of the genome of the tubercle bacillus, Mycobacterium tuberculosis, was constructed by using a twin-pronged approach. Pulsed-field gel electrophoretic analysis enabled cleavage sites for Asn I and Dra I to be positioned on the 4.4-Mb circular chromosome, while, in parallel, clones from two cosmid libraries were ordered into contigs by means of fingerprinting and hybridization mapping. The resultant contig map was readily correlated with the physical map of the genome via the landmarked restriction sites. Over 165 genes and markers were localized on the integrated map, thus enabling comparisons with the leprosy bacillus, Mycobacterium leprae, to be undertaken. Mycobacterial genomes appear to have evolved as mosaic structures since extended segments with conserved gene order and organization are interspersed with different flanking regions. Repetitive sequences and insertion elements are highly abundant in M. tuberculosis, but the distribution of IS6110 is apparently nonrandom. Images Fig. 1 Fig. 2 PMID:8610181

  20. Pulmonary Disease due to Mycobacterium tuberculosis in a Horse: Zoonotic Concerns and Limitations of Antemortem Testing

    PubMed Central

    Lyashchenko, Konstantin P.; Greenwald, Rena; Esfandiari, Javan; Lecu, Alexis; Waters, W. Ray; Posthaus, Horst; Bodmer, Thomas; Janssens, Jean-Paul; Aloisio, Fabio; Graubner, Claudia; Grosclaude, Eléonore; Piersigilli, Alessandra; Schiller, Irene

    2012-01-01

    A case of pulmonary tuberculosis caused by Mycobacterium tuberculosis was diagnosed in a horse. Clinical evaluation performed prior to euthanasia did not suggest tuberculosis, but postmortem examination provided pathological and bacteriological evidence of mycobacteriosis. In the lungs, multiple tuberculoid granulomas communicating with the bronchiolar lumen, pleural effusion, and a granulomatous lymphadenitis involving mediastinal and tracheobronchial lymph nodes were found. Serologic response to M. tuberculosis antigens was detected in the infected horse, but not in the group of 42 potentially exposed animals (18 horses, 14 alpacas, 6 donkeys, and 4 dogs) which showed no signs of disease. Diagnosis of tuberculosis in live horses remains extremely difficult. Four of 20 animal handlers at the farm were positive for tuberculous infection upon follow-up testing by interferon-gamma release assay, indicating a possibility of interspecies transmission of M. tuberculosis. PMID:22567544

  1. Pulmonary Disease due to Mycobacterium tuberculosis in a Horse: Zoonotic Concerns and Limitations of Antemortem Testing.

    PubMed

    Lyashchenko, Konstantin P; Greenwald, Rena; Esfandiari, Javan; Lecu, Alexis; Waters, W Ray; Posthaus, Horst; Bodmer, Thomas; Janssens, Jean-Paul; Aloisio, Fabio; Graubner, Claudia; Grosclaude, Eléonore; Piersigilli, Alessandra; Schiller, Irene

    2012-01-01

    A case of pulmonary tuberculosis caused by Mycobacterium tuberculosis was diagnosed in a horse. Clinical evaluation performed prior to euthanasia did not suggest tuberculosis, but postmortem examination provided pathological and bacteriological evidence of mycobacteriosis. In the lungs, multiple tuberculoid granulomas communicating with the bronchiolar lumen, pleural effusion, and a granulomatous lymphadenitis involving mediastinal and tracheobronchial lymph nodes were found. Serologic response to M. tuberculosis antigens was detected in the infected horse, but not in the group of 42 potentially exposed animals (18 horses, 14 alpacas, 6 donkeys, and 4 dogs) which showed no signs of disease. Diagnosis of tuberculosis in live horses remains extremely difficult. Four of 20 animal handlers at the farm were positive for tuberculous infection upon follow-up testing by interferon-gamma release assay, indicating a possibility of interspecies transmission of M. tuberculosis. PMID:22567544

  2. Mycobacterium tuberculosis isolates from single outpatient clinic in Panama City exhibit wide genetic diversity.

    PubMed

    Sambrano, Dilcia; Correa, Ricardo; Almengor, Pedro; Domínguez, Amada; Vega, Silvio; Goodridge, Amador

    2014-08-01

    Understanding Mycobacterium tuberculosis biodiversity and transmission is significant for tuberculosis control. This short report aimed to determine the genetic diversity of M. tuberculosis isolates from an outpatient clinic in Panama City. A total of 62 M. tuberculosis isolates were genotyped by 12 loci mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) and Spoligotyping. Forty-five (72.6%) of the isolates showed unique MIRU-VNTR genotypes, and 13 (21%) of the isolates were grouped into four clusters. Four isolates showed polyclonal MIRU-VNTR genotypes. The MIRU-VNTR Hunter-Gaston discriminatory index reached 0.988. The Spoligotyping analysis revealed 16 M. tuberculosis families, including Latin American-Mediterranean, Harlem, and Beijing. These findings suggest a wide genetic diversity of M. tuberculosis isolates at one outpatient clinic. A detailed molecular epidemiology survey is now warranted, especially following second massive immigration for local Panama Canal expansion activities. PMID:24865686

  3. Human Exposure following Mycobacterium tuberculosis Infection of Multiple Animal Species in a Metropolitan Zoo

    PubMed Central

    Oh, Peter; Granich, Reuben; Scott, Jim; Sun, Ben; Joseph, Michael; Stringfield, Cynthia; Thisdell, Susan; Staley, Jothan; Workman-Malcolm, Donna; Borenstein, Lee; Lehnkering, Eleanor; Ryan, Patrick; Soukup, Jeanne; Nitta, Annette

    2002-01-01

    From 1997 to 2000, Mycobacterium tuberculosis was diagnosed in two Asian elephants (Elephas maximus), three Rocky Mountain goats (Oreamnos americanus), and one black rhinoceros (Diceros bicornis) in the Los Angeles Zoo. DNA fingerprint patterns suggested recent transmission. An investigation found no active cases of tuberculosis in humans; however, tuberculin skin-test conversions in humans were associated with training elephants and attending an elephant necropsy. PMID:12453358

  4. Specificity of IS6110-based amplification assays for Mycobacterium tuberculosis complex.

    PubMed Central

    Hellyer, T J; DesJardin, L E; Assaf, M K; Bates, J H; Cave, M D; Eisenach, K D

    1996-01-01

    The specificity of IS6110 for the Mycobacterium tuberculosis complex has recently been questioned. We observed no cross-reaction with 27 nontuberculous mycobacteria using strand displacement- and PCR-based amplification of the nucleotide 970 to 1026 and 762 to 865 regions of IS6110. These data support use of selected regions of IS6110 as M. tuberculosis complex-specific targets. PMID:8897197

  5. Rifampin Heteroresistance in Mycobacterium tuberculosis Cultures as Detected by Phenotypic and Genotypic Drug Susceptibility Test Methods

    PubMed Central

    Folkvardsen, Dorte Bek; Thomsen, Vibeke Ø.; Rigouts, Leen; Rasmussen, Erik Michael; Bang, Didi; Bernaerts, Gertjan; Werngren, Jim; Toro, Juan Carlos; Hoffner, Sven; Hillemann, Doris

    2013-01-01

    Tuberculosis patients may harbor both drug-susceptible and -resistant bacteria, i.e., heteroresistance. We used mixtures of rifampin-resistant and -susceptible Mycobacterium tuberculosis strains to simulate heteroresistance in patient samples. Molecular tests can be used for earlier discovery of multidrug resistance (MDR), but the sensitivity to detect heteroresistance is unknown. Conventional phenotypic drug susceptibility testing was the most sensitive, whereas two line probe assays and sequencing were unable to detect the clinically important 1% resistant bacteria. PMID:24068005

  6. Antagonistic action of Streptococcus salivarius and Streptococcus faecalis to Mycobacterium tuberculosis.

    PubMed Central

    Darling, C L; Hart, G D

    1976-01-01

    Streptococcus salivarius and Streptococcus faecalis were found to inhibit the growth of Mycobacterium tuberculosis on Löwenstein-Jensen and Middlebrook 7H11 agars, but not on the latter medium when antibacterial drugs were added. S. faecalis was found to be more inhibitory than S. salivarius to 15 strains of M. tuberculosis. S. salivarius produced little or no inhibition of growth of Runyon group III organisms but was very antagonistic to Runyon group I mycobacteria. Images PMID:824304

  7. IFNG-mediated immune responses enhance autophagy against Mycobacterium tuberculosis antigens in patients with active tuberculosis

    PubMed Central

    Rovetta, Ana I; Peña, Delfina; Hernández Del Pino, Rodrigo E; Recalde, Gabriela M; Pellegrini, Joaquín; Bigi, Fabiana; Musella, Rosa M; Palmero, Domingo J; Gutierrez, Marisa; Colombo, María I; García, Verónica E

    2015-01-01

    Protective immunity against Mycobacterium tuberculosis (Mtb) requires IFNG. Besides, IFNG-mediated induction of autophagy suppresses survival of virulent Mtb in macrophage cell lines. We investigated the contribution of autophagy to the defense against Mtb antigen (Mtb-Ag) in cells from tuberculosis patients and healthy donors (HD). Patients were classified as high responders (HR) if their T cells produced significant IFNG against Mtb-Ag; and low responders (LR) when patients showed weak or no T cell responses to Mtb-Ag. The highest autophagy levels were detected in HD cells whereas the lowest quantities were observed in LR patients. Interestingly, upon Mtb-Ag stimulation, we detected a positive correlation between IFNG and MAP1LC3B-II/LC3-II levels. Actually, blockage of Mtb-Ag-induced IFNG markedly reduced autophagy in HR patients whereas addition of limited amounts of IFNG significantly increased autophagy in LR patients. Therefore, autophagy collaborates with human immune responses against Mtb in close association with specific IFNG secreted against the pathogen. PMID:25426782

  8. Potential Inhibitors for Isocitrate Lyase of Mycobacterium tuberculosis and Non-M. tuberculosis: A Summary

    PubMed Central

    Lee, Yie-Vern; Wahab, Habibah A.

    2015-01-01

    Isocitrate lyase (ICL) is the first enzyme involved in glyoxylate cycle. Many plants and microorganisms are relying on glyoxylate cycle enzymes to survive upon downregulation of tricarboxylic acid cycle (TCA cycle), especially Mycobacterium tuberculosis (MTB). In fact, ICL is a potential drug target for MTB in dormancy. With the urge for new antitubercular drug to overcome tuberculosis treat such as multidrug resistant strain and HIV-coinfection, the pace of drug discovery has to be increased. There are many approaches to discovering potential inhibitor for MTB ICL and we hereby review the updated list of them. The potential inhibitors can be either a natural compound or synthetic compound. Moreover, these compounds are not necessary to be discovered only from MTB ICL, as it can also be discovered by a non-MTB ICL. Our review is categorized into four sections, namely, (a) MTB ICL with natural compounds; (b) MTB ICL with synthetic compounds; (c) non-MTB ICL with natural compounds; and (d) non-MTB ICL with synthetic compounds. Each of the approaches is capable of overcoming different challenges of inhibitor discovery. We hope that this paper will benefit the discovery of better inhibitor for ICL. PMID:25649791

  9. Potential inhibitors for isocitrate lyase of Mycobacterium tuberculosis and non-M. tuberculosis: a summary.

    PubMed

    Lee, Yie-Vern; Wahab, Habibah A; Choong, Yee Siew

    2015-01-01

    Isocitrate lyase (ICL) is the first enzyme involved in glyoxylate cycle. Many plants and microorganisms are relying on glyoxylate cycle enzymes to survive upon downregulation of tricarboxylic acid cycle (TCA cycle), especially Mycobacterium tuberculosis (MTB). In fact, ICL is a potential drug target for MTB in dormancy. With the urge for new antitubercular drug to overcome tuberculosis treat such as multidrug resistant strain and HIV-coinfection, the pace of drug discovery has to be increased. There are many approaches to discovering potential inhibitor for MTB ICL and we hereby review the updated list of them. The potential inhibitors can be either a natural compound or synthetic compound. Moreover, these compounds are not necessary to be discovered only from MTB ICL, as it can also be discovered by a non-MTB ICL. Our review is categorized into four sections, namely, (a) MTB ICL with natural compounds; (b) MTB ICL with synthetic compounds; (c) non-MTB ICL with natural compounds; and (d) non-MTB ICL with synthetic compounds. Each of the approaches is capable of overcoming different challenges of inhibitor discovery. We hope that this paper will benefit the discovery of better inhibitor for ICL. PMID:25649791

  10. Sublineages of Mycobacterium tuberculosis Beijing genotype strains and unfavorable outcomes of anti-tuberculosis treatment.

    PubMed

    Hang, Nguyen T L; Maeda, Shinji; Keicho, Naoto; Thuong, Pham H; Endo, Hiroyoshi

    2015-05-01

    The influence of Mycobacterium tuberculosis (MTB) lineages/sublineages on unfavorable tuberculosis (TB) treatment outcomes is poorly understood. We investigated the effects of Beijing genotype sublineages and other factors contributing to treatment outcome. Patients newly diagnosed with sputum smear-positive and culture-positive TB in Hanoi, Vietnam, participated in the study. After receiving anti-TB treatment, they were intensively followed up for the next 16 months. MTB isolates collected before treatment were subjected to drug susceptibility testing, and further analyzed to determine MTB (sub) lineages and their clonal similarities. Of 430 patients, 17 had treatment failure and 30 had TB recurrence. Rifampicin resistance was associated with treatment failure {adjusted odds ratio = 6.64 [95% confidence interval (CI), 1.48-29.73]}. The modern Beijing genotype was significantly associated with recurrent TB within 16 months [adjusted hazard ratio = 3.29 (95% CI, 1.17-9.27)], particularly after adjustment for the relevant antibiotic resistance. Human immunodeficiency virus coinfection and severity on chest radiographs were not significantly associated with unfavorable outcomes. Our findings provide further understanding of the influence of MTB strains on unfavorable treatment outcomes. Multiple risk factors should be considered for the optimal management of TB. PMID:25732626

  11. Role of Fused Mycobacterium tuberculosis Immunogens and Adjuvants in Modern Tuberculosis Vaccines.

    PubMed

    Junqueira-Kipnis, Ana Paula; Marques Neto, Lázaro Moreira; Kipnis, André

    2014-01-01

    Several approaches have been developed to improve or replace the only available vaccine for tuberculosis (TB), BCG (Bacille Calmette Guerin). The development of subunit protein vaccines is a promising strategy because it combines specificity and safety. In addition, subunit protein vaccines can be designed to have selected immune epitopes associated with immunomodulating components to drive the appropriate immune response. However, the limited antigens present in subunit vaccines reduce their capacity to stimulate a complete immune response compared with vaccines composed of live attenuated or killed microorganisms. This deficiency can be compensated by the incorporation of adjuvants in the vaccine formulation. The fusion of adjuvants with Mycobacterium tuberculosis (Mtb) proteins or immune epitopes has the potential to become the new frontier in the TB vaccine development field. Researchers have addressed this approach by fusing the immune epitopes of their vaccines with molecules such as interleukins, lipids, lipoproteins, and immune stimulatory peptides, which have the potential to enhance the immune response. The fused molecules are being tested as subunit vaccines alone or within live attenuated vector contexts. Therefore, the objectives of this review are to discuss the association of Mtb fusion proteins with adjuvants; Mtb immunogens fused with adjuvants; and cytokine fusion with Mtb proteins and live recombinant vectors expressing cytokines. The incorporation of adjuvant molecules in a vaccine can be complex, and developing a stable fusion with proteins is a challenging task. Overall, the fusion of adjuvants with Mtb epitopes, despite the limited number of studies, is a promising field in vaccine development. PMID:24795730

  12. Molecular Typing of Mycobacterium Tuberculosis Strains: A Fundamental Tool for Tuberculosis Control and Elimination

    PubMed Central

    Cannas, Angela; Mazzarelli, Antonio; Di Caro, Antonino; Delogu, Giovanni; Girardi, Enrico

    2016-01-01

    Tuberculosis (TB) is still an important cause of morbidity and mortality worldwide. An improvement of the strategies for disease control is necessary in both low- and high-incidence TB countries. Clinicians, epidemiologists, laboratory specialists, and public health players should work together in order to achieve a significant reduction in TB transmission and spread of drug-resistant strains. Effective TB surveillance relies on early diagnosis of new cases, appropriate therapy, and accurate detection of outbreaks in the community, in order to implement proper TB control strategies. To achieve this goal, information from classical and molecular epidemiology, together with patient clinical data need to be combined. In this review, we summarize the methodologies currently used in molecular epidemiology, namely molecular typing. We will discuss their efficiency to phylogenetically characterize Mycobacterium tuberculosis isolates, and their ability to provide information that can be useful for disease control. We will also introduce next generation sequencing as the methodology that potentially could provide in a short time both, detection of new outbreaks and identification of resistance patterns. This could envision a potential of next generation sequencing as an important tool for accurate patient management and disease control. PMID:27403266

  13. Comparative analysis of mycobacterium and related actinomycetes yields insight into the evolution of mycobacterium tuberculosis pathogenesis

    PubMed Central

    2012-01-01

    Background The sequence of the pathogen Mycobacterium tuberculosis (Mtb) strain H37Rv has been available for over a decade, but the biology of the pathogen remains poorly understood. Genome sequences from other Mtb strains and closely related bacteria present an opportunity to apply the power of comparative genomics to understand the evolution of Mtb pathogenesis. We conducted a comparative analysis using 31 genomes from the Tuberculosis Database (TBDB.org), including 8 strains of Mtb and M. bovis, 11 additional Mycobacteria, 4 Corynebacteria, 2 Streptomyces, Rhodococcus jostii RHA1, Nocardia farcinia, Acidothermus cellulolyticus, Rhodobacter sphaeroides, Propionibacterium acnes, and Bifidobacterium longum. Results Our results highlight the functional importance of lipid metabolism and its regulation, and reveal variation between the evolutionary profiles of genes implicated in saturated and unsaturated fatty acid metabolism. It also suggests that DNA repair and molybdopterin cofactors are important in pathogenic Mycobacteria. By analyzing sequence conservation and gene expression data, we identify nearly 400 conserved noncoding regions. These include 37 predicted promoter regulatory motifs, of which 14 correspond to previously validated motifs, as well as 50 potential noncoding RNAs, of which we experimentally confirm the expression of four. Conclusions Our analysis of protein evolution highlights gene families that are associated with the adaptation of environmental Mycobacteria to obligate pathogenesis. These families include fatty acid metabolism, DNA repair, and molybdopterin biosynthesis. Our analysis reinforces recent findings suggesting that small noncoding RNAs are more common in Mycobacteria than previously expected. Our data provide a foundation for understanding the genome and biology of Mtb in a comparative context, and are available online and through TBDB.org. PMID:22452820

  14. Heme Oxygenase-1 Regulates Inflammation and Mycobacterial Survival in Human Macrophages during Mycobacterium tuberculosis Infection.

    PubMed

    Scharn, Caitlyn R; Collins, Angela C; Nair, Vidhya R; Stamm, Chelsea E; Marciano, Denise K; Graviss, Edward A; Shiloh, Michael U

    2016-06-01

    Mycobacterium tuberculosis, the causative agent of tuberculosis, is responsible for 1.5 million deaths annually. We previously showed that M. tuberculosis infection in mice induces expression of the CO-producing enzyme heme oxygenase (HO1) and that CO is sensed by M. tuberculosis to initiate a dormancy program. Further, mice deficient in HO1 succumb to M. tuberculosis infection more readily than do wild-type mice. Although mouse macrophages control intracellular M. tuberculosis infection through several mechanisms, such as NO synthase, the respiratory burst, acidification, and autophagy, how human macrophages control M. tuberculosis infection remains less well understood. In this article, we show that M. tuberculosis induces and colocalizes with HO1 in both mouse and human tuberculosis lesions in vivo, and that M. tuberculosis induces and colocalizes with HO1 during primary human macrophage infection in vitro. Surprisingly, we find that chemical inhibition of HO1 both reduces inflammatory cytokine production by human macrophages and restricts intracellular growth of mycobacteria. Thus, induction of HO1 by M. tuberculosis infection may be a mycobacterial virulence mechanism to enhance inflammation and bacterial growth. PMID:27183573

  15. Profiling Antibodies to Mycobacterium tuberculosis by Multiplex Microbead Suspension Arrays for Serodiagnosis of Tuberculosis▿

    PubMed Central

    Khan, Imran H.; Ravindran, Resmi; Yee, JoAnn; Ziman, Melanie; Lewinsohn, David M.; Gennaro, Marila L.; Flynn, JoAnne L.; Goulding, Celia W.; DeRiemer, Kathryn; Lerche, Nickolas W.; Luciw, Paul A.

    2008-01-01

    Tuberculosis (TB) is a serious global disease. The fatality rate attributed to TB is among the highest of infectious diseases, with approximately 2 million deaths occurring per year worldwide. Identification of individuals infected with Mycobacterium tuberculosis and screening of their immediate contacts is crucial for controlling the spread of TB. Current methods for detection of M. tuberculosis infection are not efficient, in particular, for testing large numbers of samples. We report a novel and efficient multiplex microbead immunoassay (MMIA), based on Luminex technology, for profiling antibodies to M. tuberculosis. Microbead sets identifiable by unique fluorescence were individually coated with each of several M. tuberculosis antigens and tested in multiplex format for antibody detection in the experimental nonhuman primate model of TB. Certain M. tuberculosis antigens, e.g., ESAT-6, CFP-10, and HspX, were included to enhance the specificity of the MMIA, because these antigens are absent in nontuberculous mycobacteria and the vaccine strain Mycobacterium bovis bacillus Calmette-Guérin. The MMIA enabled simultaneous detection of multiple M. tuberculosis plasma antibodies in several cohorts of macaques representing different stages of infection and/or disease. Antibody profiles were defined in early and latent/chronic infection. These proof-of-concept findings demonstrate the potential clinical use of the MMIA. In addition, the MMIA serodetection system has a potential for mining M. tuberculosis open reading frames (about 4,000) to discover novel target proteins for the development of more-comprehensive TB serodiagnostic tests. PMID:18077619

  16. A Chlorhexidine- Agar Plate Culture Medium Protocol to Complement Standard Broth Culture of Mycobacterium tuberculosis

    PubMed Central

    Asmar, Shady; Chatellier, Sonia; Mirande, Caroline; van Belkum, Alex; Canard, Isabelle; Raoult, Didier; Drancourt, Michel

    2016-01-01

    The culture of Mycobacterium tuberculosis using parallel inoculation of a solid culture medium and a liquid broth provides the gold standard for the diagnosis of tuberculosis. Here, we evaluated a chlorhexidine decontamination-MOD9 solid medium protocol versus the standard NALC-NaOH-Bactec 960 MGIT protocol for the diagnosis of pulmonary tuberculosis by culture. Three-hundred clinical specimens comprising 193 sputa, 30 bronchial aspirates, 10 broncho-alveolar lavages, 47 stools, and 20 urines were prospectively submitted for the routine diagnosis of tuberculosis. The contamination rates were 5/300 (1.7%) using the MOD9 protocol and 17/300 (5.7%) with the Bactec protocol, respectively (P < 0.05, Fisher exact test). Of a total of 50 Mycobacterium isolates (48 M. tuberculosis and two Mycobacterium abscessus) were cultured. Out of these 50, 48 (96%) isolates were found using the MOD9 protocol versus 35 (70%) when using the Bactec protocol (P < 0.05, Fisher exact test). The time to positivity was 10.1 ± 3.9 days versus 14.7 ± 7.3 days, respectively, (P < 0.05, Student’s t-test). These data confirmed the usefulness of parallel inoculation of a solid culture medium with broth for the recovery of M. tuberculosis in agreement with current recommendations. More specifically, chlorhexidine decontamination and inoculation of the MOD9 solid medium could be proposed to complement the standard Bactec 960 MGIT broth protocol. PMID:26834733

  17. A Chlorhexidine- Agar Plate Culture Medium Protocol to Complement Standard Broth Culture of Mycobacterium tuberculosis.

    PubMed

    Asmar, Shady; Chatellier, Sonia; Mirande, Caroline; van Belkum, Alex; Canard, Isabelle; Raoult, Didier; Drancourt, Michel

    2016-01-01

    The culture of Mycobacterium tuberculosis using parallel inoculation of a solid culture medium and a liquid broth provides the gold standard for the diagnosis of tuberculosis. Here, we evaluated a chlorhexidine decontamination-MOD9 solid medium protocol versus the standard NALC-NaOH-Bactec 960 MGIT protocol for the diagnosis of pulmonary tuberculosis by culture. Three-hundred clinical specimens comprising 193 sputa, 30 bronchial aspirates, 10 broncho-alveolar lavages, 47 stools, and 20 urines were prospectively submitted for the routine diagnosis of tuberculosis. The contamination rates were 5/300 (1.7%) using the MOD9 protocol and 17/300 (5.7%) with the Bactec protocol, respectively (P < 0.05, Fisher exact test). Of a total of 50 Mycobacterium isolates (48 M. tuberculosis and two Mycobacterium abscessus) were cultured. Out of these 50, 48 (96%) isolates were found using the MOD9 protocol versus 35 (70%) when using the Bactec protocol (P < 0.05, Fisher exact test). The time to positivity was 10.1 ± 3.9 days versus 14.7 ± 7.3 days, respectively, (P < 0.05, Student's t-test). These data confirmed the usefulness of parallel inoculation of a solid culture medium with broth for the recovery of M. tuberculosis in agreement with current recommendations. More specifically, chlorhexidine decontamination and inoculation of the MOD9 solid medium could be proposed to complement the standard Bactec 960 MGIT broth protocol. PMID:26834733

  18. Streptomycin-Starved Mycobacterium tuberculosis 18b, a Drug Discovery Tool for Latent Tuberculosis

    PubMed Central

    Zhang, Ming; Sala, Claudia; Hartkoorn, Ruben C.; Dhar, Neeraj; Mendoza-Losana, Alfonso

    2012-01-01

    Mycobacterium tuberculosis 18b, a streptomycin (STR)-dependent mutant that enters a viable but nonreplicating state in the absence of STR, has been developed as a simple model for drug testing against dormant bacilli. Here, we further evaluated the STR-starved 18b (SS18b) model both in vitro and in vivo by comparing the behavior of 22 approved and experimental tuberculosis drugs. Using the resazurin reduction microplate assay (REMA), rifampin (RIF), rifapentine (RPT), TMC207, clofazimine (CFM), and linezolid (LIN) were found to be active against SS18b in vitro, and their bactericidal activity was confirmed by determining the number of CFU. A latent 18b infection was established in mice, and some of the above-mentioned drugs were used for treatment, either alone or in combination with RIF. RIF, RPT, TMC207, CFM, and pyrazinamide (PZA) were all active in vivo, while cell wall inhibitors were not. A comparative kinetic study of rifamycin efficacy was then undertaken, and the results indicated that RPT clears latent 18b infection in mice faster than RIF. Intrigued by the opposing responses of live and dormant 18b cells to cell wall inhibitors, we conducted a systematic analysis of 14 such inhibitors using REMA. This uncovered an SS18b signature (CWPRED) that accurately predicted the activities of cell wall inhibitors and performed well in a blind study. CWPRED will be useful for establishing the mode of action of compounds with unknown targets, while the SS18b system should facilitate the discovery of drugs for treating latent tuberculosis. PMID:22926567

  19. Protection by novel vaccine candidates, Mycobacterium tuberculosis ΔmosR and ΔechA7, against challenge with a Mycobacterium tuberculosis Beijing strain.

    PubMed

    Marcus, Sarah A; Steinberg, Howard; Talaat, Adel M

    2015-10-13

    Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), infects over two billion people, claiming around 1.5 million lives annually. The only vaccine approved for clinical use against this disease is the Bacillus Calmette-Guérin (BCG) vaccine. Unfortunately, BCG has limited efficacy against the adult, pulmonary form of tuberculosis. This vaccine was developed from M. bovis with antigen expression and host specificity that differ from M. tuberculosis. To address these problems, we have designed two novel, live attenuated vaccine (LAV) candidates on an M. tuberculosis background: ΔmosR and ΔechA7. These targeted genes are important to M. tuberculosis pathogenicity during infection. To examine the efficacy of these strains, C57BL/6 mice were vaccinated subcutaneously with either LAV, BCG, or PBS. Both LAV strains persisted up to 16 weeks in the spleens or lungs of vaccinated mice, while eliciting minimal pathology prior to challenge. Following challenge with a selected, high virulence M. tuberculosis Beijing strain, protection was notably greater for both groups of LAV vaccinated animals as compared to BCG at both 30 and 60 days post-challenge. Additionally, vaccination with either ΔmosR or ΔechA7 elicited an immune response similar to BCG. Although these strains require further development to meet safety standards, this first evidence of protection by these two new, live attenuated vaccine candidates shows promise. PMID:26363381

  20. Biochemical characterization of recombinant phosphoglucose isomerase of Mycobacterium tuberculosis

    SciTech Connect

    Mathur, Divya; Ahsan, Zaid; Tiwari, Madhulika; Garg, Lalit C. . E-mail: lalitcgarg@yahoo.com

    2005-11-18

    Phosphoglucose isomerase (PGI) is a well-characterized ubiquitous enzyme involved in the glycolytic pathway. It catalyzes the reversible isomerization of D-glucopyranose-6-phosphate and D-fructofuranose-6-phosphate and is present in all living cells. However, there is interspecies variation at the level of the primary structure which sometimes produces heterogeneity at the structural and functional levels. In order to evaluate and characterize the mycobacterial PGI, the gene encoding the PGI from Mycobacterium tuberculosis H37Rv was cloned in pET-22b(+) vector and expressed in Escherichia coli. The target DNA was PCR amplified from the bacterial artificial chromosome using specific primers and cloned under the control of T7 promoter. Upon induction with IPTG, the recombinant PGI (rPGI) expressed partly as soluble protein and partly as inclusion bodies. The rPGI from the soluble fraction was purified to near homogeneity by ion-exchange chromatography. Mass spectrum analysis of the purified rPGI revealed its mass to be 61.45 kDa. The purified rPGI was enzymatically active and the specific activity was 600 U/mg protein. The K {sub m} of rPGI was determined to be 0.318 mM for fructose-6-phosphate and the K {sub i} was 0.8 mM for 6-phosphogluconate. The rPGI exhibited optimal activity at 37 deg C and pH 9.0, and did not require mono- or divalent cations for its activity.

  1. Catalysis and Inhibition of Mycobacterium tuberculosis Methionine Aminopeptidase

    SciTech Connect

    Lu, Jing-Ping; Chai, Sergio C.; Ye, Qi-Zhuang

    2010-09-07

    Methionine aminopeptidase (MetAP) carries out an important cotranslational N-terminal methionine excision of nascent proteins and represents a potential target to develop antibacterial and antitubercular drugs. We cloned one of the two MetAPs in Mycobacterium tuberculosis (MtMetAP1c from the mapB gene) and purified it to homogeneity as an apoenzyme. Its activity required a divalent metal ion, and Co(II), Ni(II), Mn(II), and Fe(II) were among activators of the enzyme. Co(II) and Fe(II) had the tightest binding, while Ni(II) was the most efficient cofactor for the catalysis. MtMetAP1c was also functional in E. coli cells because a plasmid-expressed MtMetAP1c complemented the essential function of MetAP in E. coli and supported the cell growth. A set of potent MtMetAP1c inhibitors were identified, and they showed high selectivity toward the Fe(II)-form, the Mn(II)-form, or the Co(II) and Ni(II) forms of the enzyme, respectively. These metalloform selective inhibitors were used to assign the metalloform of the cellular MtMetAP1c. The fact that only the Fe(II)-form selective inhibitors inhibited the cellular MtMetAP1c activity and inhibited the MtMetAP1c-complemented cell growth suggests that Fe(II) is the native metal used by MtMetAP1c in an E. coli cellular environment. Finally, X-ray structures of MtMetAP1c in complex with three metalloform-selective inhibitors were analyzed, which showed different binding modes and different interactions with metal ions and active site residues.

  2. Diagnostic Accuracy of Intracellular Mycobacterium tuberculosis Detection for Tuberculous Meningitis

    PubMed Central

    Feng, Guo-dong; Shi, Ming; Ma, Lei; Chen, Ping; Wang, Bing-ju; Zhang, Min; Chang, Xiao-lin; Su, Xiu-chu; Yang, Yi-ning; Fan, Xin-hong; Dai, Wen; Liu, Ting-ting; He, Ying; Bian, Ting; Duan, Li-xin; Li, Jin-ge; Hao, Xiao-ke; Liu, Jia-yun; Xue, Xin; Song, Yun-zhang; Wu, Hai-qin; Niu, Guo-qiang; Zhang, Li; Han, Cui-juan; Lin, Hong; Lin, Zhi-hui; Liu, Jian-jun; Jian, Qian; Zhang, Jin-she; Tian, Ye; Zhou, Bai-yu; Wang, Jing; Xue, Chang-hu; Han, Xiao-fang; Wang, Jian-feng; Wang, Shou-lian

    2014-01-01

    Rationale: Early diagnosis and treatment of tuberculous meningitis saves lives, but current laboratory diagnostic tests lack sensitivity. Objectives: We investigated whether the detection of intracellular bacteria by a modified Ziehl-Neelsen stain and early secretory antigen target (ESAT)-6 in cerebrospinal fluid leukocytes improves tuberculous meningitis diagnosis. Methods: Cerebrospinal fluid specimens from patients with suspected tuberculous meningitis were stained by conventional Ziehl-Neelsen stain, a modified Ziehl-Neelsen stain involving cytospin slides with Triton processing, and an ESAT-6 immunocytochemical stain. Acid-fast bacteria and ESAT-6–expressing leukocytes were detected by microscopy. All tests were performed prospectively in a central laboratory by experienced technicians masked to the patients’ final diagnosis. Measurements and Main Results: Two hundred and eighty patients with suspected tuberculous meningitis were enrolled. Thirty-seven had Mycobacterium tuberculosis cultured from cerebrospinal fluid; 40 had a microbiologically confirmed alternative diagnosis; the rest had probable or possible tuberculous meningitis according to published criteria. Against a clinical diagnostic gold standard the sensitivity of conventional Ziehl-Neelsen stain was 3.3% (95% confidence interval, 1.6–6.7%), compared with 82.9% (95% confidence interval, 77.4–87.3%) for modified Ziehl-Neelsen stain and 75.1% (95% confidence interval, 68.8–80.6%) for ESAT-6 immunostain. Intracellular bacteria were seen in 87.8% of the slides positive by the modified Ziehl-Neelsen stain. The specificity of modified Ziehl-Neelsen and ESAT-6 stain was 85.0% (95% confidence interval, 69.4–93.8%) and 90.0% (95% confidence interval, 75.4–96.7%), respectively. Conclusions: Enhanced bacterial detection by simple modification of the Ziehl-Neelsen stain and an ESAT-6 intracellular stain improve the laboratory diagnosis of tuberculous meningitis. PMID:24450377

  3. Epidemiologic surveillance to detect false-positive Mycobacterium tuberculosis cultures.

    PubMed

    Lee, Meng-Rui; Chung, Kuei-Pin; Chen, Wei-Ting; Huang, Yu-Tsung; Lee, Li-Na; Yu, Chong-Jen; Teng, Lee-Jene; Hsueh, Po-Ren; Yang, Pan-Chyr; Luh, Kwen-Tay

    2012-08-01

    This study was aimed to investigate the ability of potential indices from epidemiologic surveillance to detect false-positive cultures of Mycobacterium tuberculosis (MTB). All clinical specimens for mycobacterial culture from April 1 to August 31, 2010, were reviewed. Single-positive cultures without relevant clinical and pathologic information were categorized as suspected false-positive cultures. Genotyping methods were used to confirm false-positive cultures. The performance of epidemiologic surveillance indices to detect potential false-positive cultures was evaluated. A total of 14,462 specimens were sent to the laboratory and 214 batches were processed in 107 work days (average 67.6 specimens per batch, ranging from 21 to 130 specimens per batch). Seventy-one single-positive cultures were identified, among which 5 cultures of multidrug-resistant MTB in 1 batch were false-positive, confirmed by genotyping methods. Epidemiologic surveillance with statistical process control charts for single-positive cultures per day showed good performance in epidemiologic surveillance. The false-positive rate was 38.5% in the 13 potential false-positive cultures according to the statistical process control chart for single-positive cultures per day. Although the incidence of tuberculous disease is high in Taiwan, clustering of multidrug-resistant MTB in 1 batch or clustering of single-positive cultures still suggested the occurrence of false-positive MTB cultures. Therefore, epidemiologic surveillance for the clustering of single-positive cultures with the statistical process control chart could be used to monitor the occurrence of false-positive results. PMID:22705229

  4. Detection of Mycobacterium tuberculosis by PCR amplification with pan-Mycobacterium primers and hybridization to an M. tuberculosis-specific probe.

    PubMed Central

    Tevere, V J; Hewitt, P L; Dare, A; Hocknell, P; Keen, A; Spadoro, J P; Young, K K

    1996-01-01

    Nucleic acid amplification techniques such as the PCR are very useful in the rapid diagnosis of infections by Mycobacterium tuberculosis. However, recent studies have shown that the accuracy of results can vary widely when tests are performed with nonstandardized reagents. We have developed a PCR assay for the detection of M. tuberculosis that is both rapid and accurate. The assay reagents are standardized and quality controlled. False-positive results due to carryover contamination are prevented by the incorporation of dUTP coupled with uracil-N-glycosylase restriction. This assay also employs pan-Mycobacterium amplification primers, allowing for flexibility in the mycobacterial species that can be identified from a single amplification reaction. The amplification is very sensitive; amplification products generated from as few as three bacteria can be detected by agarose gel electrophoresis. DNAs isolated from 33 of 34 mycobacterial species tested were amplified efficiently. Only DNA from Mycobacterium simiae did not amplify. The amplification is also very specific. Amplification products were generated only from the DNAs of bacteria in closely related genera such as Corynebacterium. The nonmycobacterial amplicons do not pose a problem, as they do not hybridize to mycobacterium-specific probes. Hybridization of amplicons to an M. tuberculosis-specific probe allows for the unambiguous identification of M. tuberculosis complex organisms. The clinical performance of this PCR assay was evaluated against that of culture in 662 respiratory specimens. Sensitivities of 100 and 73.1% were obtained from smear-positive and -negative respiratory specimens, respectively. The corresponding specificities were 100 and 99.8%. The high sensitivity and specificity, coupled with the potential for detecting a wide range of mycobacteria, make this assay a useful tool in the clinical management of mycobacterial infections. PMID:8815108

  5. Microarray analysis of Mycobacterium tuberculosis-infected monocytes reveals IL26 as a new candidate gene for tuberculosis susceptibility

    PubMed Central

    Guerra-Laso, José M; Raposo-García, Sara; García-García, Silvia; Diez-Tascón, Cristina; Rivero-Lezcano, Octavio M

    2015-01-01

    Differences in the activity of monocytes/macrophages, important target cells of Mycobacterium tuberculosis, might influence tuberculosis progression. With the purpose of identifying candidate genes for tuberculosis susceptibility we infected monocytes from both healthy elderly individuals (a tuberculosis susceptibility group) and elderly tuberculosis patients with M. tuberculosis, and performed a microarray experiment. We detected 78 differentially expressed transcripts and confirmed these results by quantitative PCR of selected genes. We found that monocytes from tuberculosis patients showed similar expression patterns for these genes, regardless of whether they were obtained from younger or older patients. Only one of the detected genes corresponded to a cytokine: IL26, a member of the interleukin-10 (IL-10) cytokine family which we found to be down-regulated in infected monocytes from tuberculosis patients. Non-infected monocytes secreted IL-26 constitutively but they reacted strongly to M. tuberculosis infection by decreasing IL-26 production. Furthermore, IL-26 serum concentrations appeared to be lower in the tuberculosis patients. When whole blood was infected, IL-26 inhibited the observed pathogen-killing capability. Although lymphocytes expressed IL26R, the receptor mRNA was not detected in either monocytes or neutrophils, suggesting that the inhibition of anti-mycobacterial activity may be mediated by lymphocytes. Additionally, IL-2 concentrations in infected blood were lower in the presence of IL-26. The negative influence of IL-26 on the anti-mycobacterial activity and its constitutive presence in both serum and monocyte supernatants prompt us to propose IL26 as a candidate gene for tuberculosis susceptibility. PMID:25157980

  6. [Occupational exposure to Mycobacterium tuberculosis complex: state of the art and prospects].

    PubMed

    Verso, M G; Caracappa, S; Vitale, F; Vesco, G; Picciotto, D

    2002-01-01

    Tuberculosis, disease never wiped out in the world, is still actual for many reasons, such as imposing emigration from poor to industrialized countries, spreading of Mycobacteria drug-resistant and presence of AIDS sick subjects. Estimation of world cases seems to under-value real number; moreover physicians consider as etiological agent only Mycobacterium tuberculosis, disregarding others Mycobacteria, such as M. bovis, giving rise a disease similar to that caused by M. tuberculosis. To single out all these variants, it's necessary to use very specific and sensitive diagnostic methods, such as Polymerase Chain Reaction, evaluating also possible work exposure, especially when occurs contacts with infectious animals. PMID:12161953

  7. Viral Booster Vaccines Improve Mycobacterium bovis BCG-Induced Protection Against Bovine Tuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Previous work in small animal laboratory models of tuberculosis have shown that vaccination strategies based on heterologous prime-boost protocols using Mycobacterium bovis bacille Calmette-Guerin (BCG) to prime and Modified Vaccinia Ankara strain (MVA85A) or recombinant attenuated adenoviruses (Ad8...

  8. Intrafamilial cluster of pulmonary tuberculosis due to Mycobacterium bovis of the African 1 clonal complex.

    PubMed

    Godreuil, S; Jeziorski, E; Bañuls, A L; Fraisse, T; Van de Perre, P; Boschiroli, M L

    2010-12-01

    A new clonal complex of Mycobacterium bovis present at high frequency in cattle from west central African countries has been described as the African 1 (Af1) clonal complex. Here, the first intrafamilial cluster of human tuberculosis cases due to M. bovis Af1 clonal complex strains is reported. We discuss hypotheses regarding modes of transmission. PMID:20980573

  9. 78 FR 36698 - Microbiology Devices; Reclassification of Nucleic Acid-Based Systems for Mycobacterium tuberculosis

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-06-19

    ... Nucleic Acid-Based Systems for Mycobacterium tuberculosis Complex in Respiratory Specimens AGENCY: Food...) is proposing to reclassify nucleic acid-based in vitro diagnostic devices for the detection of... Controls Guideline: Nucleic Acid-Based In Vitro Diagnostic Devices for the Detection of...

  10. GSMN-TB: a web-based genome-scale network model of Mycobacterium tuberculosis metabolism

    PubMed Central

    Beste, Dany JV; Hooper, Tracy; Stewart, Graham; Bonde, Bhushan; Avignone-Rossa, Claudio; Bushell, Michael E; Wheeler, Paul; Klamt, Steffen; Kierzek, Andrzej M; McFadden, Johnjoe

    2007-01-01

    Background An impediment to the rational development of novel drugs against tuberculosis (TB) is a general paucity of knowledge concerning the metabolism of Mycobacterium tuberculosis, particularly during infection. Constraint-based modeling provides a novel approach to investigating microbial metabolism but has not yet been applied to genome-scale modeling of M. tuberculosis. Results GSMN-TB, a genome-scale metabolic model of M. tuberculosis, was constructed, consisting of 849 unique reactions and 739 metabolites, and involving 726 genes. The model was calibrated by growing Mycobacterium bovis bacille Calmette Guérin in continuous culture and steady-state growth parameters were measured. Flux balance analysis was used to calculate substrate consumption rates, which were shown to correspond closely to experimentally determined values. Predictions of gene essentiality were also made by flux balance analysis simulation and were compared with global mutagenesis data for M. tuberculosis grown in vitro. A prediction accuracy of 78% was achieved. Known drug targets were predicted to be essential by the model. The model demonstrated a potential role for the enzyme isocitrate lyase during the slow growth of mycobacteria, and this hypothesis was experimentally verified. An interactive web-based version of the model is available. Conclusion The GSMN-TB model successfully simulated many of the growth properties of M. tuberculosis. The model provides a means to examine the metabolic flexibility of bacteria and predict the phenotype of mutants, and it highlights previously unexplored features of M. tuberculosis metabolism. PMID:17521419

  11. Tuberculosis in Sudan: a study of Mycobacterium tuberculosis strain genotype and susceptibility to anti-tuberculosis drugs

    PubMed Central

    2011-01-01

    Background Sudan is a large country with a diverse population and history of civil conflict. Poverty levels are high with a gross national income per capita of less than two thousand dollars. The country has a high burden of tuberculosis (TB) with an estimated 50,000 incident cases during 2009, when the estimated prevalence was 209 cases per 100,000 of the population. Few studies have been undertaken on TB in Sudan and the prevalence of drug resistant disease is not known. Methods In this study Mycobacterium tuberculosis isolates from 235 patients attending three treatment centers in Sudan were screened for susceptibility to isoniazid, rifampicin, ethambutol and streptomycin by the proportion method on Lowenstein Jensen media. 232 isolates were also genotyped by spoligotyping. Demographic details of patients were recorded using a structured questionnaire. Statistical analyses were conducted to examine the associations between drug resistance with risk ratios computed for a set of risk factors (gender, age, case status - new or relapse, geographic origin of the patient, spoligotype, number of people per room, marital status and type of housing). Results Multi drug-resistant tuberculosis (MDR-TB), being resistance to at least rifampicin and isoniazid, was found in 5% (95% CI: 2,8) of new cases and 24% (95% CI: 14,34) of previously treated patients. Drug resistance was associated with previous treatment with risk ratios of 3.51 (95% CI: 2.69-4.60; p < 0.001) for resistance to any drug and 5.23 (95% CI: 2.30-11.90; p < 0.001) for MDR-TB. Resistance was also associated with the geographic region of origin of the patient, being most frequently observed in patients from the Northern region and least in the Eastern region with risk ratios of 7.43 (95%CI:3.42,16.18; p: < 0.001) and 14.09 (95%CI:1.80,110.53; p:0.026) for resistance to any drug and MDR-TB. The major genotype observed was of the Central Asia spoligotype family (CAS1_Delhi), representing 49% of the 232 isolates

  12. Rapid discrimination of Mycobacterium tuberculosis strains by random amplified polymorphic DNA analysis.

    PubMed Central

    Linton, C J; Jalal, H; Leeming, J P; Millar, M R

    1994-01-01

    Investigations of the epidemiology of tuberculosis have been hampered by the lack of strain-specific markers that can be used to differentiate isolates of Mycobacterium tuberculosis. We report the development of a rapid protocol for random amplified polymorphic DNA analysis which included the use of a commercially available DNA extraction kit (GeneReleaser). This was applied to 14 strains of M. tuberculosis, including strains associated with temporal and geographical clusters of tuberculosis in the United Kingdom and those from India, Africa, and Saudi Arabia. Strains of M. tuberculosis could be discriminated in about 8 h by this method, which is therefore a rapid and simple alternative to restriction fragment length polymorphism analysis. Images PMID:7814542

  13. The Importance of First Impressions: Early Events in Mycobacterium tuberculosis Infection Influence Outcome

    PubMed Central

    Cadena, Anthony M.; Fortune, Sarah M.

    2016-01-01

    ABSTRACT Tuberculosis remains a major health threat in much of the world. New vaccines against Mycobacterium tuberculosis are essential for preventing infection, disease, and transmission. However, the host immune responses that need to be induced by an effective vaccine remain unclear. Increasingly, it has become clear that early events in infection are of major importance in the eventual outcome of the infection. Studying such events in humans is challenging, as they occur within the lung and thoracic lymph nodes, and any clinical signs of early infection are relatively nonspecific. Nonetheless, clinical studies and animal models of tuberculosis have provided new insights into the local events that occur in the first few weeks of tuberculosis. Development of an effective vaccine requires a clear understanding of the successful (and detrimental) early host responses against M. tuberculosis, with the goal to improve upon natural immune responses and prevent infection or disease. PMID:27048801

  14. MicroRNA-365 in macrophages regulates Mycobacterium tuberculosis-induced active pulmonary tuberculosis via interleukin-6.

    PubMed

    Song, Qingzhang; Li, Hui; Shao, Hua; Li, Chunling; Lu, Xiao

    2015-01-01

    The present study is to investigate the relationship between microRNA (miR)-365 expression and the levels of interleukin (IL)-6 mRNA and protein in patients with active tuberculosis. From June 2011 to June 2014, 48 patients with active pulmonary tuberculosis induced by Mycobacterium tuberculosis were included in the study. In addition, 23 healthy subjects were enrolled as control. Macrophages were collected by pulmonary alveolus lavage. In addition, serum and mononuclear cells were isolated from peripheral blood. The levels of miR-365 and IL-6 in macrophages, mononuclear cells and serum were determined using quantitative real-time polymerase chain reaction. The protein expression of IL-6 in macrophages and mononuclear cells was measured using Western blotting, while that in serum was detected by enzyme-linked immunoabsorbent assay. Expression of IL-6 mRNA and protein was significantly enhanced in patients with active pulmonary tuberculosis. Increase of IL-6 protein concentration in serum was probably due to the release of IL-6 protein from mononuclear cells in the blood. In addition, miR-365 levels were significantly lowered in patients with active pulmonary tuberculosis. Up-regulated IL-6 expression in macrophages, mononuclear cells and serum in patients with active pulmonary tuberculosis is related to the down-regulation of miR-365, suggesting that miR-365 may regulate the occurrence and immune responses of active pulmonary tuberculosis via IL-6. PMID:26629035

  15. MicroRNA-365 in macrophages regulates Mycobacterium tuberculosis-induced active pulmonary tuberculosis via interleukin-6

    PubMed Central

    Song, Qingzhang; Li, Hui; Shao, Hua; Li, Chunling; Lu, Xiao

    2015-01-01

    The present study is to investigate the relationship between microRNA (miR)-365 expression and the levels of interleukin (IL)-6 mRNA and protein in patients with active tuberculosis. From June 2011 to June 2014, 48 patients with active pulmonary tuberculosis induced by Mycobacterium tuberculosis were included in the study. In addition, 23 healthy subjects were enrolled as control. Macrophages were collected by pulmonary alveolus lavage. In addition, serum and mononuclear cells were isolated from peripheral blood. The levels of miR-365 and IL-6 in macrophages, mononuclear cells and serum were determined using quantitative real-time polymerase chain reaction. The protein expression of IL-6 in macrophages and mononuclear cells was measured using Western blotting, while that in serum was detected by enzyme-linked immunoabsorbent assay. Expression of IL-6 mRNA and protein was significantly enhanced in patients with active pulmonary tuberculosis. Increase of IL-6 protein concentration in serum was probably due to the release of IL-6 protein from mononuclear cells in the blood. In addition, miR-365 levels were significantly lowered in patients with active pulmonary tuberculosis. Up-regulated IL-6 expression in macrophages, mononuclear cells and serum in patients with active pulmonary tuberculosis is related to the down-regulation of miR-365, suggesting that miR-365 may regulate the occurrence and immune responses of active pulmonary tuberculosis via IL-6. PMID:26629035

  16. First-Line Anti-Tubercular Drug Resistance of Mycobacterium tuberculosis in IRAN: A Systematic Review

    PubMed Central

    Pourakbari, Babak; Mamishi, Setareh; Mohammadzadeh, Mona; Mahmoudi, Shima

    2016-01-01

    Background: The spread of drug-resistant tuberculosis (TB) is one of the major public health problems through the world. Surveillance of anti-TB drug resistance is essential for monitoring of TB control strategies. The occurrence of drug resistance, particularly multi-drug resistance Mycobacterium tuberculosis (MDR), defined as resistance to at least rifampicin (RIF) and isoniazid (INH), has become a significant public health dilemma. The status of drug-resistance TB in Iran, one of the eastern Mediterranean countries locating between Azerbaijan and Armenia and high-TB burden countries (such as Afghanistan and Pakistan) has been reported inconsistently. Therefore, the aim of this study was to summarize reports of first-line anti-tubercular drug resistance in M. tuberculosis in Iran. Material and Methods: We systematically reviewed published studies on drug-resistant M. tuberculosis in Iran. The search terms were “Mycobacterium tuberculosis susceptibility” or “Mycobacterium tuberculosis resistant” and Iran. Results: Fifty-two eligible articles, published during 1998–2014, were included in this review. Most of the studies were conducted in Tehran. The most common used laboratory method for detecting M. tuberculosis drug resistant was Agar proportion. The highest resistance to first-line drugs was seen in Tehran, the capital city of Iran. The average prevalence of isoniazid (INH), rifampin (RIF), streptomycin (SM), and ethambotol (EMB) resistance via Agar proportion method in Tehran was 26, 23, 22.5, and 16%, respectively. In general, resistance to INH was more common than RIF, SM, and EMB in Tehran Conclusions: In conclusion, this systematic review summarized the prevalence and distribution of first-line anti-tubercular drug resistance of M. tuberculosis in Iran. Our results suggested that effective strategies to minimize the acquired drug resistance, to control the transmission of resistance and improve the diagnosis measures for TB control in Iran. PMID

  17. Pupylation-dependent and -independent proteasomal degradation in mycobacteria.

    PubMed

    Imkamp, Frank; Ziemski, Michal; Weber-Ban, Eilika

    2015-08-01

    Bacteria make use of compartmentalizing protease complexes, similar in architecture but not homologous to the eukaryotic proteasome, for the selective and processive removal of proteins. Mycobacteria as members of the actinobacteria harbor proteasomes in addition to the canonical bacterial degradation complexes. Mycobacterial proteasomal degradation, although not essential during normal growth, becomes critical for survival under particular environmental conditions, like, for example, during persistence of the pathogenic Mycobacterium tuberculosis in host macrophages or of environmental mycobacteria under starvation. Recruitment of protein substrates for proteasomal degradation is usually mediated by pupylation, the post-translational modification of lysine side chains with the prokaryotic ubiquitin-like protein Pup. This substrate recruitment strategy is functionally reminiscent of ubiquitination in eukaryotes, but is the result of convergent evolution, relying on chemically and structurally distinct enzymes. Pupylated substrates are recognized by the ATP-dependent proteasomal regulator Mpa that associates with the 20S proteasome core. A pupylation-independent proteasome degradation pathway has recently been discovered that is mediated by the ATP-independent bacterial proteasome activator Bpa (also referred to as PafE), and that appears to play a role under stress conditions. In this review, mechanistic principles of bacterial proteasomal degradation are discussed and compared with functionally related elements of the eukaryotic ubiquitin-proteasome system. Special attention is given to an understanding on the molecular level based on structural and biochemical analysis. Wherever available, discussion of in vivo studies is included to highlight the biological significance of this unusual bacterial degradation pathway. PMID:26352358

  18. Mycobacterium tuberculosis evolutionary pathogenesis and its putative impact on drug development.

    PubMed

    Le Chevalier, Fabien; Cascioferro, Alessandro; Majlessi, Laleh; Herrmann, Jean Louis; Brosch, Roland

    2014-01-01

    Mycobacterium tuberculosis, the etiological agent of human TB, is the most important mycobacterial pathogen in terms of global patient numbers and gravity of disease. The molecular mechanisms by which M. tuberculosis causes disease are complex and the result of host-pathogen coevolution that might have started already in the time of its Mycobacterium canettii-like progenitors. Despite research progress, M. tuberculosis still holds many secrets of its successful strategy for circumventing host defences, persisting in the host and developing resistance, which makes anti-TB treatment regimens extremely long and often inefficient. Here, we discuss what we have learned from recent studies on the evolution of the pathogen and its putative new drug targets that are essential for mycobacterial growth under in vitro or in vivo conditions. PMID:25302954

  19. Multidrug-Resistant Mycobacterium tuberculosis of the Latin American Mediterranean Lineage, Wrongly Identified as Mycobacterium pinnipedii (Spoligotype International Type 863 [SIT863]), Causing Active Tuberculosis in South Brazil

    PubMed Central

    Vasconcelos, Sidra E. G.; Esteves, Leonardo S.; Gomes, Harrison M.; Almeida da Silva, Pedro; Perdigão, João; Portugal, Isabel; Viveiros, Miguel; McNerney, Ruth; Pain, Arnab; Clark, Taane G.; Rastogi, Nalin; Unis, Gisela; Rossetti, Maria Lucia R.

    2015-01-01

    We recently detected the spoligotype patterns of strains of Mycobacterium pinnipedii, a species of the Mycobacterium tuberculosis complex, in sputum samples from nine cases with pulmonary tuberculosis residing in Porto Alegre, South Brazil. Because this species is rarely encountered in humans, we further characterized these nine isolates by additional genotyping techniques, including 24-locus mycobacterial interspersed repetitive-unit–variable-number tandem-repeat (MIRU-VNTR) typing, verification of the loci TbD1, RD9, pks15/1, RDRio, and fbpC, the insertion of IS6110 at a site specific to the M. tuberculosis Latin American Mediterranean (LAM) lineage, and whole-genome sequencing. The combined analysis of these markers revealed that the isolates are in fact M. tuberculosis and more specifically belong to the LAM genotype. Most of these isolates (n = 8) were shown to be multidrug resistant (MDR), which prompted us to perform partial sequencing of the rpoA, rpoB, rpoC, katG, and inhA genes. Seven isolates (77.8%) carried the S315T mutation in katG, and one of these (11%) also presented the C(−17)T single-nucleotide polymorphism (SNP) in inhA. Interestingly, six of the MDR isolates also presented an undescribed insertion of 12 nucleotides (CCA GAA CAA CCC) in codon 516 of rpoB. No putative compensatory mutation was found in either rpoA or rpoC. This is the first report of an M. tuberculosis LAM family strain with a convergent M. pinnipedii spoligotype. These spoligotypes are observed in genotype databases at a modest frequency, highlighting that care must be taken when identifying isolates in the M. tuberculosis complex on the basis of single genetic markers. PMID:26400784

  20. Comparison of the Construction of Unmarked Deletion Mutations in Mycobacterium smegmatis, Mycobacterium bovis Bacillus Calmette-Guérin, and Mycobacterium tuberculosis H37Rv by Allelic Exchange

    PubMed Central

    Pavelka, Martin S.; Jacobs, William R.

    1999-01-01

    Until recently, genetic analysis of Mycobacterium tuberculosis, the causative agent of tuberculosis, was hindered by a lack of methods for gene disruptions and allelic exchange. Several groups have described different methods for disrupting genes marked with antibiotic resistance determinants in the slow-growing organisms Mycobacterium bovis bacillus Calmette-Guérin (BCG) and M. tuberculosis. In this study, we described the first report of using a mycobacterial suicidal plasmid bearing the counterselectable marker sacB for the allelic exchange of unmarked deletion mutations in the chromosomes of two substrains of M. bovis BCG and M. tuberculosis H37Rv. In addition, our comparison of the recombination frequencies in these two slow-growing species and that of the fast-growing organism Mycobacterium smegmatis suggests that the homologous recombination machinery of the three species is equally efficient. The mutants constructed here have deletions in the lysA gene, encoding meso-diaminopimelate decarboxylase, an enzyme catalyzing the last step in lysine biosynthesis. We observed striking differences in the lysine auxotrophic phenotypes of these three species of mycobacteria. The M. smegmatis mutant can grow on lysine-supplemented defined medium or complex rich medium, while the BCG mutants grow only on lysine-supplemented defined medium and are unable to form colonies on complex rich medium. The M. tuberculosis lysine auxotroph requires 25-fold more lysine on defined medium than do the other mutants and is dependent upon the detergent Tween 80. The mutants described in this work are potential vaccine candidates and can also be used for studies of cell wall biosynthesis and amino acid metabolism. PMID:10438745

  1. Phenotypically Adapted Mycobacterium tuberculosis Populations from Sputum Are Tolerant to First-Line Drugs

    PubMed Central

    Turapov, Obolbek; O'Connor, Benjamin D.; Sarybaeva, Asel A.; Williams, Caroline; Patel, Hemu; Kadyrov, Abdullaat S.; Sarybaev, Akpay S.; Woltmann, Gerrit; Barer, Michael R.

    2016-01-01

    Tuberculous sputum contains multiple Mycobacterium tuberculosis populations with different requirements for isolation in vitro. These include cells that form colonies on solid media (plateable M. tuberculosis), cells requiring standard liquid medium for growth (nonplateable M. tuberculosis), and cells requiring supplementation of liquid medium with culture supernatant (SN) for growth (SN-dependent M. tuberculosis). Here, we describe protocols for the cryopreservation and direct assessment of antimicrobial tolerance of these M. tuberculosis populations within sputum. Our results show that first-line drugs achieved only modest bactericidal effects on all three populations over 7 days (1 to 2.5 log10 reductions), and SN-dependent M. tuberculosis was more tolerant to streptomycin and isoniazid than the plateable and nonplateable M. tuberculosis strains. Susceptibility of plateable M. tuberculosis to bactericidal drugs was significantly increased after passage in vitro; thus, tolerance observed in the sputum samples from the population groups was likely associated with mycobacterial adaptation to the host environment at some time prior to expectoration. Our findings support the use of a simple ex vivo system for testing drug efficacies against mycobacteria that have phenotypically adapted during tuberculosis infection. PMID:26883695

  2. Revisiting the role of phospholipases C in virulence and the lifecycle of Mycobacterium tuberculosis

    PubMed Central

    Le Chevalier, Fabien; Cascioferro, Alessandro; Frigui, Wafa; Pawlik, Alexandre; Boritsch, Eva C.; Bottai, Daria; Majlessi, Laleh; Herrmann, Jean Louis; Brosch, Roland

    2015-01-01

    Mycobacterium tuberculosis, the agent of human tuberculosis has developed different virulence mechanisms and virulence-associated tools during its evolution to survive and multiply inside the host. Based on previous reports and by analogy with other bacteria, phospholipases C (PLC) of M. tuberculosis were thought to be among these tools. To get deeper insights into the function of PLCs, we investigated their putative involvement in the intracellular lifestyle of M. tuberculosis, with emphasis on phagosomal rupture and virulence, thereby re-visiting a research theme of longstanding interest. Through the construction and use of an M. tuberculosis H37Rv PLC-null mutant (ΔPLC) and control strains, we found that PLCs of M. tuberculosis were not required for induction of phagosomal rupture and only showed marginal, if any, impact on virulence of M. tuberculosis in the cellular and mouse infection models used in this study. In contrast, we found that PLC-encoding genes were strongly upregulated under phosphate starvation and that PLC-proficient M. tuberculosis strains survived better than ΔPLC mutants under conditions where phosphatidylcholine served as sole phosphate source, opening new perspectives for studies on the role of PLCs in the lifecycle of M. tuberculosis. PMID:26603639

  3. Risk factors for Mycobacterium tuberculosis infection among children in Greenland

    PubMed Central

    Andersen, Aase Bengaard; Melbye, Mads; Wohlfahrt, Jan; Andersson, Mikael; Biggar, Robert J; Ladefoged, Karin; Thomsen, Vibeke Ostergaard; Koch, Anders

    2011-01-01

    Abstract Objective To examine the risk factors for Mycobacterium tuberculosis infection (MTI) among Greenlandic children for the purpose of identifying those at highest risk of infection. Methods Between 2005 and 2007, 1797 Greenlandic schoolchildren in five different areas were tested for MTI with an interferon gamma release assay (IGRA) and a tuberculin skin test (TST). Parents or guardians were surveyed using a standardized self-administered questionnaire to obtain data on crowding in the household, parents’ educational level and the child’s health status. Demographic data for each child – i.e. parents’ place of birth, number of siblings, distance between siblings (next younger and next older), birth order and mother’s age when the child was born – were also extracted from a public registry. Logistic regression was used to check for associations between these variables and MTI, and all results were expressed as odds ratios (ORs) and 95% confidence intervals (CIs). Children were considered to have MTI if they tested positive on both the IGRA assay and the TST. Findings The overall prevalence of MTI was 8.5% (152/1797). MTI was diagnosed in 26.7% of the children with a known TB contact, as opposed to 6.4% of the children without such contact. Overall, the MTI rate was higher among Inuit children (OR: 4.22; 95% CI: 1.55–11.5) and among children born less than one year after the birth of the next older sibling (OR: 2.48; 95% CI: 1.33–4.63). Self-reported TB contact modified the profile to include household crowding and low mother’s education. Children who had an older MTI-positive sibling were much more likely to test positive for MTI themselves (OR: 14.2; 95% CI: 5.75–35.0) than children without an infected older sibling. Conclusion Ethnicity, sibling relations, number of household residents and maternal level of education are factors associated with the risk of TB infection among children in Greenland. The strong household clustering of

  4. The quiet and controversial: Ural family of Mycobacterium tuberculosis.

    PubMed

    Mokrousov, Igor

    2012-06-01

    The absence of lateral gene exchange is a characteristic feature defining the genome evolution and clonal population structure of Mycobacterium tuberculosis. Certain of its lineages have justly attracted more attention due to their global dissemination and/or remarkable pathogenic properties. In this critical review, I discuss the population structure and genetic geography of the less 'popular' but in some aspects no less noteworthy M. tuberculosis lineage, Ural family. Its specific signature was initially defined by single copy in MIRU26, and large (>6) copy number in MIRU10 loci, and by 43-spoligotyping as absence of signals 29-31 and 33-36. Here, I suggest to subdivide Ural strains with present and absent spoligosignal 2 into primary Ural-1 and secondary Ural-2 sublineages, respectively, while 1 copy in MIRU26 is specific of Ural-1. Furthermore, three copies were recently described in MIRU10 in Ural-1 strains which highlights a high diversity of this locus in Ural genotype. The data on the two Ural sublineages were extracted from SpolDB4 database and original publications in order to trace their distribution at global and within-country levels. Importantly, the rigorous reanalysis suggested the true rate of the Ural genotype in the Ural area in Russia to be only 7%. In contrast, the frequencies of the Ural sublineages peak elsewhere: in South Ukraine and Georgia/Abkhazia (Ural-1, up to 14-19%), and in southwestern Iran (Ural-2, up to 26%). However, as this name is used since 2005, it seems most parsimonious to continue its use even if misleading. The forest graph was built on the available spoligoprofiles of Ural family strains from Eurasia. It helped to suggest routes of their primary dispersal that are discussed in the context of the known human migrations also influenced by natural barriers. The north/east Pontic area may have been an area of origin and primary dispersal of the Ural (Ural-1) genotype in Eurasia, whereas political and natural borders may have

  5. Validation of a real-time PCR assay for the molecular identification of Mycobacterium tuberculosis

    PubMed Central

    Sales, Mariana L.; Fonseca, Antônio Augusto; Orzil, Lívia; Alencar, Andrea Padilha; Silva, Marcio Roberto; Issa, Marina Azevedo; Filho, Paulo Martins Soares; Lage, Andrey Pereira; Heinemann, Marcos Bryan

    2014-01-01

    Mycobacterium tuberculosis is the major cause of tuberculosis in humans. This bacillus gained prominence with the occurrence of HIV, presenting itself as an important opportunistic infection associated with acquired immunodeficiency syndrome (AIDS). The current study aimed to develop a real-time PCR using Eva Green technology for molecular identification of M. tuberculosis isolates. The primers were designed to Rv1510 gene. Ninety nine samples of M. tuberculosis and sixty samples of M. bovis were tested and no sample of the bovine bacillus was detected by the qPCR. Statistical tests showed no difference between the qPCR and biochemical tests used to identify the Mycobacterium tuberculosis. The correlation between tests was perfect with Kappa index of 1.0 (p < 0.001, CI = 0.84 – 1.0). The diagnostic sensitivity and specificity were 100% (CI = 95.94% – 100%) and 100% (CI = 93.98% – 100%). This qPCR was developed with the goal of diagnosing the bacillus M. tuberculosis in samples of bacterial suspension. TB reference laboratories (health and agriculture sectors), public health programs and epidemiological studies probably may benefit from such method. PMID:25763042

  6. Orchestration of pulmonary T cell immunity during Mycobacterium tuberculosis infection: immunity interruptus

    PubMed Central

    Behar, Samuel M.; Carpenter, Stephen M.; Booty, Matthew G.; Barber, Daniel L.; Jayaraman, Pushpa

    2014-01-01

    Despite the introduction almost a century ago of Mycobacterium bovis BCG (BCG), an attenuated form of M. bovis that is used as a vaccine against Mycobacterium tuberculosis, tuberculosis remains a global health threat and kills more than 1.5 million people each year. This is mostly because BCG fails to prevent pulmonary disease – the contagious form of tuberculosis. Although there have been significant advances in understanding how the immune system responds to infection, the qualities that define protective immunity against M. tuberculosis remain poorly characterized. The ability to predict who will maintain control over the infection and who will succumb to clinical disease would revolutionize our approach to surveillance, control, and treatment. Here we review the current understanding of pulmonary T cell responses following M. tuberculosis infection. While infection elicits a strong immune response that contains infection, M. tuberculosis evades eradication. Traditionally, its intracellular lifestyle and alteration of macrophage function are viewed as the dominant mechanisms of evasion. Now we appreciate that chronic inflammation leads to T cell dysfunction. While this may arise as the host balances the goals of bacterial sterilization and avoidance of tissue damage, it is becoming clear that T cell dysfunction impairs host resistance. Defining the mechanisms that lead to T cell dysfunction is crucial as memory T cell responses are likely to be subject to the same subject to the same pressures. Thus, success of T cell based vaccines is predicated on memory T cells avoiding exhaustion while at the same time not promoting overt tissue damage. PMID:25311810

  7. Detection of Mycobacterium tuberculosis complex by nested polymerase chain reaction in pulmonary and extrapulmonary specimens* ,**

    PubMed Central

    Furini, Adriana Antônia da Cruz; Pedro, Heloisa da Silveira Paro; Rodrigues, Jean Francisco; Montenegro, Lilian Maria Lapa; Machado, Ricardo Luiz Dantas; Franco, Célia; Schindler, Haiana Charifker; Batista, Ida Maria Foschiani Dias; Rossit, Andrea Regina Baptista

    2013-01-01

    OBJECTIVE: To compare the performance of nested polymerase chain reaction (NPCR) with that of cultures in the detection of the Mycobacterium tuberculosis complex in pulmonary and extrapulmonary specimens. METHODS: We analyzed 20 and 78 pulmonary and extrapulmonary specimens, respectively, of 67 hospitalized patients suspected of having tuberculosis. An automated microbial system was used for the identification of Mycobacterium spp. cultures, and M. tuberculosis IS6110 was used as the target sequence in the NPCR. The kappa statistic was used in order to assess the level of agreement among the results. RESULTS: Among the 67 patients, 6 and 5, respectively, were diagnosed with pulmonary and extrapulmonary tuberculosis, and the NPCR was positive in all of the cases. Among the 98 clinical specimens, smear microscopy, culture, and NPCR were positive in 6.00%, 8.16%, and 13.26%, respectively. Comparing the results of NPCR with those of cultures (the gold standard), we found that NPCR had a sensitivity and specificity of 100% and 83%, respectively, in pulmonary specimens, compared with 83% and 96%, respectively, in extrapulmonary specimens, with good concordance between the tests (kappa, 0.50 and 0.6867, respectively). CONCLUSIONS: Although NPCR proved to be a very useful tool for the detection of M. tuberculosis complex, clinical, epidemiological, and other laboratory data should also be considered in the diagnosis and treatment of pulmonary and extrapulmonary tuberculosis. PMID:24473765

  8. MspA-Mycobacterium tuberculosis-transformant with reduced virulence: the "unbirthday paradigm".

    PubMed

    Lamrabet, Otmane; Ghigo, Eric; Mège, Jean-Louis; Lepidi, Hubert; Nappez, Claude; Raoult, Didier; Drancourt, Michel

    2014-11-01

    Expressing mspA porin gene from Mycobacterium smegmatis in Mycobacterium tuberculosis attenuated this pathogen. Intracellular growth of the transformants into free-living amoeba and murine and human macrophages decreased. Furthermore, transformants decreased the microbicidal program of human monocyte-derived macrophages. BALB/c mice inoculated with transformants exhibited higher weights, lower histological lesions and lower M. tuberculosis inoculum in the liver, spleen and lungs than control mice challenged with wild-type M. tuberculosis. Preliminary evaluation indicated that mice inoculated with this transformant showed higher weights and lower numbers of lung nodules and tissular mycobacteria than control mice when challenged with wild-type M. tuberculosis. Similar to the paradoxical "unbirthday" gift coined by Lewis Carroll in Alice's Adventures in Wonderland, adding mspA gene reduced the virulence of M. tuberculosis and yielded a protective effect. Lost of non-virulence genes is a mechanism for virulence in mycobacteria. Engineering non-virulence genes in M. tuberculosis may yield strains with decreased virulence and increased immunogenicity. PMID:25194334

  9. In Vitro and In Vivo Activities of the Nitroimidazole TBA-354 against Mycobacterium tuberculosis

    PubMed Central

    Cho, S.; Yang, T. J.; Kim, Y.; Wang, Y.; Lu, Y.; Wang, B.; Xu, J.; Mdluli, K.; Ma, Z.; Franzblau, S. G.

    2014-01-01

    Nitroimidazoles are a promising new class of antitubercular agents. The nitroimidazo-oxazole delamanid (OPC-67683, Deltyba) is in phase III trials for the treatment of multidrug-resistant tuberculosis, while the nitroimidazo-oxazine PA-824 is entering phase III for drug-sensitive and drug-resistant tuberculosis. TBA-354 (SN31354[(S)-2-nitro-6-((6-(4-trifluoromethoxy)phenyl)pyridine-3-yl)methoxy)-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine]) is a pyridine-containing biaryl compound with exceptional efficacy against chronic murine tuberculosis and favorable bioavailability in preliminary rodent studies. It was selected as a potential next-generation antituberculosis nitroimidazole following an extensive medicinal chemistry effort. Here, we further evaluate the pharmacokinetic properties and activity of TBA-354 against Mycobacterium tuberculosis. TBA-354 is narrow spectrum and bactericidal in vitro against replicating and nonreplicating Mycobacterium tuberculosis, with potency similar to that of delamanid and greater than that of PA-824. The addition of serum protein or albumin does not significantly alter this activity. TBA-354 maintains activity against Mycobacterium tuberculosis H37Rv isogenic monoresistant strains and clinical drug-sensitive and drug-resistant isolates. Spontaneous resistant mutants appear at a frequency of 3 × 10−7. In vitro studies and in vivo studies in mice confirm that TBA-354 has high bioavailability and a long elimination half-life. In vitro studies suggest a low risk of drug-drug interactions. Low-dose aerosol infection models of acute and chronic murine tuberculosis reveal time- and dose-dependent in vivo bactericidal activity that is at least as potent as that of delamanid and more potent than that of PA-824. Its superior potency and pharmacokinetic profile that predicts suitability for once-daily oral dosing suggest that TBA-354 be studied further for its potential as a next-generation nitroimidazole. PMID:25331696

  10. Role of Metal-Dependent Regulation of ESX-3 Secretion in Intracellular Survival of Mycobacterium tuberculosis.

    PubMed

    Tinaztepe, Emir; Wei, Jun-Rong; Raynowska, Jenelle; Portal-Celhay, Cynthia; Thompson, Victor; Philips, Jennifer A

    2016-08-01

    More people die every year from Mycobacterium tuberculosis infection than from infection by any other bacterial pathogen. Type VII secretion systems (T7SS) are used by both environmental and pathogenic mycobacteria to secrete proteins across their complex cell envelope. In the nonpathogen Mycobacterium smegmatis, the ESX-1 T7SS plays a role in conjugation, and the ESX-3 T7SS is involved in metal homeostasis. In M. tuberculosis, these secretion systems have taken on roles in virulence, and they also are targets of the host immune response. ESX-3 secretes a heterodimer composed of EsxG (TB9.8) and EsxH (TB10.4), which impairs phagosome maturation in macrophages and is essential for virulence in mice. Given the importance of EsxG and EsxH during infection, we examined their regulation. With M. tuberculosis, the secretion of EsxG and EsxH was regulated in response to iron and zinc, in accordance with the previously described transcriptional response of the esx-3 locus to these metals. While iron regulated the esx-3 expression in both M. tuberculosis and M. smegmatis, there is a significant difference in the dynamics of this regulation. In M. smegmatis, the esx-3 locus behaved like other iron-regulated genes such as mbtB In M. tuberculosis, both iron and zinc modestly repressed esx-3 expression. Diminished secretion of EsxG and EsxH in response to these metals altered the interaction of M. tuberculosis with macrophages, leading to impaired intracellular M. tuberculosis survival. Our findings detail the regulatory differences of esx-3 in M. tuberculosis and M. smegmatis and demonstrate the importance of metal-dependent regulation of ESX-3 for virulence in M. tuberculosis. PMID:27245412

  11. CD44 is a macrophage binding site for Mycobacterium tuberculosis that mediates macrophage recruitment and protective immunity against tuberculosis

    PubMed Central

    Leemans, Jaklien C.; Florquin, Sandrine; Heikens, Mirjam; Pals, Steven T.; Neut, Ronald van der; van der Poll, Tom

    2003-01-01

    Cell migration and phagocytosis are both important for controlling Mycobacterium tuberculosis infection and are critically dependent on the reorganization of the cytoskeleton. Since CD44 is an adhesion molecule involved in inflammatory responses and is connected to the actin cytoskeleton, we investigated the role of CD44 in both these processes. Macrophage (Mφ) recruitment into M. tuberculosis–infected lungs and delayed-type hypersensitivity sites was impaired in CD44-deficient (CD44–/–) mice. In addition, the number of T lymphocytes and the concentration of the protective key cytokine IFN-γ were reduced in the lungs of infected CD44–/– mice. The production of IFN-γ by splenocytes of CD44–/– mice was profoundly increased upon antigen-specific stimulation. Flow cytometry analysis revealed that soluble CD44 can directly bind to virulent M. tuberculosis. Mycobacteria also interacted with Mφ-associated CD44, as reflected by reduced binding and internalization of bacilli by CD44–/– Mφs. This suggests that CD44 is a receptor on Mφs for binding of M. tuberculosis. CD44–/– mice displayed a decreased survival and an enhanced mycobacterial outgrowth in lungs and liver during pulmonary tuberculosis. In summary, we have identified CD44 as a new Mφ binding site for M. tuberculosis that mediates mycobacterial phagocytosis, Mφ recruitment, and protective immunity against pulmonary tuberculosis. PMID:12618522

  12. Comparative analyses of the proteins from Mycobacterium tuberculosis and human genomes: Identification of potential tuberculosis drug targets.

    PubMed

    Sridhar, Settu; Dash, Pallabini; Guruprasad, Kunchur

    2016-03-15

    Tuberculosis, one of the major infectious diseases affecting human beings is caused by the bacillus Mycobacterium tuberculosis. Increased resistance to known drugs commonly used for the treatment of tuberculosis has created an urgent need to identify new targets for validation and to develop drugs. In this study, we have used various bioinformatics tools, to compare the protein sequences from twenty-three M. tuberculosis genome strains along with the known human protein sequences, in order to identify the 'conserved' M. tuberculosis proteins absent in human. Further, based on the analysis of protein interaction networks, we selected one-hundred and forty proteins that were predicted as potential M. tuberculosis drug targets and prioritized according to the ranking of 'clusters' of interacting proteins. Comparison of the predicted 140 TB targets with literature indicated that 46 of them were previously reported, thereby increasing the confidence in our predictions of the remaining 94 targets too. The analyses of the structures and functions corresponding to the predicted potential TB drug targets indicated a diverse range of proteins that included ten 'druggable' targets with some of the known drugs. PMID:26762852

  13. Insertion element IS1081-associated restriction fragment length polymorphisms in Mycobacterium tuberculosis complex species: a reliable tool for recognizing Mycobacterium bovis BCG.

    PubMed Central

    van Soolingen, D; Hermans, P W; de Haas, P E; van Embden, J D

    1992-01-01

    Recently, the insertion element IS1081 from Mycobacterium bovis was identified. In this study, the usefulness of IS1081 in the epidemiology of tuberculosis was investigated. The host range of this insertion sequence was found to be restricted exclusively to the group of Mycobacterium tuberculosis complex bacteria, whereas none of the 10 mycobacterial species which do not belong to the M. tuberculosis complex contained IS1081-homologous DNA. All 99 M. tuberculosis complex strains investigated carried five or six copies of IS1081, and very limited IS1081-associated restriction fragment length polymorphisms were observed among the strains. Seven different IS1081-containing bands were distinguished in each strain, and the patterns differed only in one or two insertion sequence-containing bands. The banding pattern of M. bovis BCG differed in the presence of a 8.0-kb IS1081-containing PvuII fragment which was absent from all other M. tuberculosis complex strains. Images PMID:1352785

  14. Identification and survival studies of Mycobacterium tuberculosis within Laboratory-Fermented bovine milk

    PubMed Central

    2014-01-01

    Background Mycobacterium tuberculosis and Mycobacterium bovis are the classic agents causing tuberculosis (TB) in humans and animals respectively. Transmission of tuberculous bacteria to humans usually occurs by inhalation of aerosols containing droplets of tubercle bacilli or via consumption of contaminated foods and drinks, primarily milk. The practice of milk pooling, including from cows with TB of the udder, further exacerbates the situation by rendering the whole milk supply infective. The simultaneous presence of indigenous lactic acid bacteria (LAB) in Mycobacterium-contaminated milk is believed to confer protective effect when the milk is adequately fermented. This study assessed the effect of LAB on the viability of mycobacteria in inherently contaminated pool of raw milk during fermentation as a function of time. Findings Growth was obtained in the pooled raw milk culture, and identified to be M. tuberculosis. This M. tuberculosis growth was undetectable in the milk culture by day 7 as assessed by plating serial dilutions of the milk culture for up to 14 days. Conclusions Some LAB species appear to show inhibitory effect on tubercle bacilli. If proven by more rigorous, controlled experimental results regarding such effect, selected LAB (with proven safety and efficacy) may have potential applications as anti-mycobacterial agents. PMID:24666844

  15. Reversal of Mycobacterium tuberculosis Phenotypic Drug Resistance by 2-Aminoimidazole Based Small Molecules

    PubMed Central

    Ackart, David F.; Lindsey, Erick A.; Podell, Brendan K.; Melander, Roberta J.; Basaraba, Randall J.; Melander, Christian

    2014-01-01

    The expression of phenotypic drug resistance or drug tolerance serves as a strategy for Mycobacterium tuberculosis to survive in vivo antimicrobial drug treatment; however the mechanisms are poorly understood. Progress toward a more in depth understanding of in vivo drug tolerance and the discovery of new therapeutic strategies designed specifically to treat drug-tolerant M. tuberculosis are hampered by the lack of appropriate in vitro assays. A library of 2-aminoimidazole based small molecules combined with the anti-tuberculosis drug isoniazid were screened against M. tuberculosis expressing in vitro drug-tolerance as microbial communities attached to an extracellular matrix derived from lysed leukocytes. Based on the ability of nine of ten 2-aminoimidazole compounds to inhibit M. smegmatis biofilm formation and three of ten molecules capable of dispersing established biofilms, two active candidates and one inactive control were tested against drug tolerant M. tuberculosis. The two active compounds restored isoniazid susceptibility as well as reduced the in vitro minimum inhibitory concentrations of isoniazid in a dose-dependent manner. The dispersion of drug tolerant M. tuberculosis with 2-aminoimidazole based small molecules as an adjunct to antimicrobial treatment has the potential to be an effective anti-tuberculosis treatment strategy designed specifically to eradicate drug-tolerant M. tuberculosis. PMID:24478046

  16. Expression of Mycobacterium tuberculosis NLPC/p60 family protein Rv0024 induce biofilm formation and resistance against cell wall acting anti-tuberculosis drugs in Mycobacterium smegmatis.

    PubMed

    Padhi, Avinash; Naik, Sumanta Kumar; Sengupta, Srabasti; Ganguli, Geetanjali; Sonawane, Avinash

    2016-04-01

    Bacterial species are capable of living as biofilm and/or planktonic forms. Role of biofilms in the pathogenesis of several human pathogens is well established. However, in case of Mycobacterium tuberculosis (Mtb) infection the role of biofilms and the genetic requirements for biofilm formation remains largely unknown. We herein report that ectopic expression of Mtb Rv0024, encoding a putative peptidoglycan amidase, in non-pathogenic Mycobacterium smegmatis(Msm) strain (MsmRv0024) confer at least 10-fold increase in resistance against two prominent anti-tuberculosis drugs isoniazid and pyrazinamide. We further report that the development of resistance was due to significant increase in biofilm formation by Rv0024. Transmission electron microscopy revealed differences in cell surface architecture of MsmRv0024 when compared with Msm wild-type (WT) and vector control Msm pSMT3 (pSMT3) strains and this aggregation pattern was due to increased cell wall hydrophobicity, as determined by Bacterial adhesion to hydrocarbons assay (BATH). Confocal microscopy study showed increased adherence of MsmRv0024 bacteria to lung epithelial cells as compared to pSMT3 strain. However, infection studies showed no differences in host cell invasion and intracellular survival in mouse macrophages. We envision that Rv0024 may play a critical role in initial infection process, adherence to host cells and drug resistance. Thus, Rv0024 may be considered as a potential drug target for the treatment of tuberculosis. PMID:26706821

  17. Detection of Mycobacteria, Mycobacterium avium Subspecies, and Mycobacterium tuberculosis Complex by a Novel Tetraplex Real-Time PCR Assay

    PubMed Central

    Molina, Elena; Elguezabal, Natalia; Pérez, Valentín; Garrido, Joseba M.

    2015-01-01

    Mycobacterium tuberculosis complex, Mycobacterium avium, and many other nontuberculous mycobacteria are worldwide distributed microorganisms of major medical and veterinary importance. Considering the growing epidemiologic significance of wildlife-livestock-human interrelation, developing rapid detection tools of high specificity and sensitivity is vital to assess their presence and accelerate the process of diagnosing mycobacteriosis. Here we describe the development and evaluation of a novel tetraplex real-time PCR for simultaneous detection of Mycobacterium genus, M. avium subspecies, and M. tuberculosis complex in an internally monitored single assay. The method was evaluated using DNA from mycobacterial (n = 38) and nonmycobacterial (n = 28) strains, tissues spiked with different CFU amounts of three mycobacterial species (n = 57), archival clinical samples (n = 233), and strains isolated from various hosts (n = 147). The minimum detectable DNA amount per reaction was 50 fg for M. bovis BCG and M. kansasii and 5 fg for M. avium subsp. hominissuis. When spiked samples were analyzed, the method consistently detected as few as 100 to 1,000 mycobacterial CFU per gram. The sensitivity and specificity values for the panel of clinical samples were 97.5 and 100% using a verified culture-based method as the reference method. The assays performed on clinical isolates confirmed these results. This PCR was able to identify M. avium and M. tuberculosis complex in the same sample in one reaction. In conclusion, the tetraplex real-time PCR we designed represents a highly specific and sensitive tool for the detection and identification of mycobacteria in routine laboratory diagnosis with potential additional uses. PMID:25588660

  18. Protection against Mycobacterium tuberculosis infection by adoptive immunotherapy. Requirement for T cell-deficient recipients

    SciTech Connect

    Orme, I.M.; Collins, F.M.

    1983-07-01

    The results of this study demonstrate that spleen cells taken from mice at the height of the primary immune response to intravenous infection with Mycobacterium tuberculosis possess the capacity to transfer adoptive protection to M. tuberculosis-infected recipients, but only if these recipients are first rendered T cell-deficient, either by thymectomy and gamma irradiation, or by sublethal irradiation. A similar requirement was necessary to demonstrate the adoptive protection of the lungs after exposure to an acute aerosol-delivered M. tuberculosis infection. In both infectious models successful adoptive immunotherapy was shown to be mediated by T lymphocytes, which were acquired in the donor animals in response to the immunizing infection. It is proposed that the results of this study may serve as a basic model for the subsequent analysis of the nature of the T cell-mediated immune response to both systemic and aerogenic infections with M. tuberculosis.

  19. Evaluation of a rapid air thermal cycler for detection of Mycobacterium tuberculosis.

    PubMed Central

    Chapin, K; Lauderdale, T L

    1997-01-01

    The Air Thermal Cycler (ATC) (Idaho Technology, Idaho Falls, Idaho) utilizes the unique technology of small-volume glass capillary tubes and high-velocity air for the heating and cooling medium for the PCR. Standard heat block thermal cycler (HBTC) and ATC performance characteristics were compared for the detection of Mycobacterium tuberculosis. Sensitivity was 100% for all smear-positive, M. tuberculosis culture-positive specimens for both the HBTC and the ATC. Of smear-negative, M. tuberculosis culture-positive specimens, sensitivity was 42.9% with the HBTC and 22.0% with the ATC. Specificity was 100% for both assay systems. Total assay time was 6.5 and 4 h and the reagent cost was 84 and 32 cents for the HBTC and ATC, respectively. The ATC offered an excellent alternative to the traditional HBTC for diagnosis of M. tuberculosis in smear-positive specimens by PCR. PMID:9230404

  20. A multi-stress model for high throughput screening against non-replicating Mycobacterium tuberculosis.

    PubMed

    Gold, Ben; Warrier, Thulasi; Nathan, Carl

    2015-01-01

    Models of non-replication help us understand the biology of persistent Mycobacterium tuberculosis. High throughput screening (HTS) against non-replicating M. tuberculosis may lead to identification of tool compounds that affect pathways on which bacterial survival depends in such states, and to development of drugs that can overcome phenotypic tolerance to conventional antimycobacterial agents, which are mostly active against replicating M. tuberculosis. We describe a multi-stress model of non-replication that mimics some of the microenvironmental conditions that M. tuberculosis faces in the host as adapted for HTS. The model includes acidic pH, mild hypoxia, a flux of nitric oxide and other reactive nitrogen intermediates arising from nitrite at low pH, and low concentrations of a fatty acid (butyrate) as a carbon source. PMID:25779324

  1. Regulation of antigen presentation by Mycobacterium tuberculosis: a role for Toll-like receptors

    PubMed Central

    Harding, Clifford V.; Boom, W. Henry

    2011-01-01

    Mycobacterium tuberculosis survives in antigen-presenting cells (APCs) such as macrophages and dendritic cells. APCs present antigens in association with major histocompatibility complex (MHC) class II molecules to stimulate CD4+ T cells, and this process is essential to contain M. tuberculosis infection. Immune evasion allows M. tuberculosis to establish persistent or latent infection in macrophages and results in Toll-like receptor 2 (TLR2)-dependent inhibition of MHC class II transactivator expression, MHC class II molecule expression and antigen presentation. This reduction of antigen presentation might reflect a general mechanism of negative-feedback regulation that prevents excessive T cell-mediated inflammation and that M. tuberculosis has subverted to create a niche for survival in infected macrophages and evasion of recognition by CD4+ T cells. PMID:20234378

  2. An Isoniazid Analogue Promotes Mycobacterium tuberculosis-Nanoparticle Interactions and Enhances Bacterial Killing by Macrophages

    PubMed Central

    de Faria, Tatiany J.; Roman, Mariane; de Souza, Nicole M.; De Vecchi, Rodrigo; de Assis, João Vitor; dos Santos, Ana Lúcia Gomes; Bechtold, Ivan H.; Winter, Nathalie; Soares, Maurilio José; Silva, Luciano Paulino; De Almeida, Mauro V.

    2012-01-01

    Nanoenabled drug delivery systems against tuberculosis (TB) are thought to control pathogen replication by targeting antibiotics to infected tissues and phagocytes. However, whether nanoparticle (NP)-based carriers directly interact with Mycobacterium tuberculosis and how such drug delivery systems induce intracellular bacterial killing by macrophages is not defined. In the present study, we demonstrated that a highly hydrophobic citral-derived isoniazid analogue, termed JVA, significantly increases nanoencapsulation and inhibits M. tuberculosis growth by enhancing intracellular drug bioavailability. Importantly, confocal and atomic force microscopy analyses revealed that JVA-NPs associate with both intracellular M. tuberculosis and cell-free bacteria, indicating that NPs directly interact with the bacterium. Taken together, these data reveal a nanotechnology-based strategy that promotes antibiotic targeting into replicating extra- and intracellular mycobacteria, which could actively enhance chemotherapy during active TB. PMID:22330919

  3. Unsuspected and extensive transmission of a drug-susceptible Mycobacterium tuberculosis strain

    PubMed Central

    López-Calleja, Ana Isabel; Gavín, Patricia; Lezcano, Ma Antonia; Vitoria, Ma Asunción; Iglesias, Ma José; Guimbao, Joaquín; Lázaro, Ma Ángeles; Rastogi, Nalin; Revillo, Ma José; Martín, Carlos; Samper, Sofia

    2009-01-01

    Background A large and unsuspected tuberculosis outbreak involving 18.7% of the total of the tuberculosis cases studied, was detected in a population-based molecular epidemiological study performed in Zaragoza (Spain) from 2001 to 2004. Methods The Mycobacterium tuberculosis drug-susceptible strain, named MTZ strain, was genetically characterized by IS6110-RFLP, Spoligotyping and by MIRU-VNTR typing and the genetic patterns obtained were compared with those included in international databases. The characteristics of the affected patients, in an attempt to understand why the MTZ strain was so highly transmitted among the population were also analyzed. Results The genetic profile of the MTZ strain was rare and not widely distributed in our area or elsewhere. The patients affected did not show any notable risk factor for TB. Conclusion The M. tuberculosis strain MTZ, might have particular transmissibility or virulence properties, and we believe that greater focus should be placed on stopping its widespread dissemination. PMID:19144198

  4. Highly structured genetic diversity of the Mycobacterium tuberculosis population in Djibouti.

    PubMed

    Godreuil, S; Renaud, F; Choisy, M; Depina, J J; Garnotel, E; Morillon, M; Van de Perre, P; Bañuls, A L

    2010-07-01

    Djibouti is an East African country with a high tuberculosis incidence. This study was conducted over a 2-month period in Djibouti, during which 62 consecutive patients with pulmonary tuberculosis (TB) were included. Genetic characterization of Mycobacterium tuberculosis, using mycobacterial interspersed repetitive-unit variable-number tandem-repeat typing and spoligotyping, was performed. The genetic and phylogenetic analysis revealed only three major families (Central Asian, East African Indian and T). The high diversity and linkage disequilibrium within each family suggest a long period of clonal evolution. A Bayesian approach shows that the phylogenetic structure observed in our sample of 62 isolates is very likely to be representative of the phylogenetic structure of the M. tuberculosis population in the total number of TB cases. PMID:19694762

  5. Integration of Published Information Into a Resistance-Associated Mutation Database for Mycobacterium tuberculosis

    PubMed Central

    Salamon, Hugh; Yamaguchi, Ken D.; Cirillo, Daniela M.; Miotto, Paolo; Schito, Marco; Posey, James; Starks, Angela M.; Niemann, Stefan; Alland, David; Hanna, Debra; Aviles, Enrique; Perkins, Mark D.; Dolinger, David L.

    2015-01-01

    Tuberculosis remains a major global public health challenge. Although incidence is decreasing, the proportion of drug-resistant cases is increasing. Technical and operational complexities prevent Mycobacterium tuberculosis drug susceptibility phenotyping in the vast majority of new and retreatment cases. The advent of molecular technologies provides an opportunity to obtain results rapidly as compared to phenotypic culture. However, correlations between genetic mutations and resistance to multiple drugs have not been systematically evaluated. Molecular testing of M. tuberculosis sampled from a typical patient continues to provide a partial picture of drug resistance. A database of phenotypic and genotypic testing results, especially where prospectively collected, could document statistically significant associations and may reveal new, predictive molecular patterns. We examine the feasibility of integrating existing molecular and phenotypic drug susceptibility data to identify associations observed across multiple studies and demonstrate potential for well-integrated M. tuberculosis mutation data to reveal actionable findings. PMID:25765106

  6. Testing chemical and genetic Modulators in Mycobacterium tuberculosis infected cells using phenotypic assays.

    PubMed

    Delorme, Vincent; Song, Ok-Ryul; Baulard, Alain; Brodin, Priscille

    2015-01-01

    Mycobacterium tuberculosis is able to colonize host cells, and it is now well admitted that the intracellular stage of the bacteria contributes to tuberculosis pathogenesis as well as to making it a persistent infection. There is still limited understanding on how the tubercle bacillus colonizes the cell and what are the factors impacting on its intracellular persistence. Recent advances in imaging technique allow rapid quantification of biological objects in complex environments. Furthermore, M. tuberculosis is a microorganism that is particularly genetically tractable and that tolerates the expression of heterologous fluorescent proteins. Thus, the intracellular distribution of M. tuberculosis expressing fluorescent proteins can be easily quantified by the use of confocal microscopy. Here we describe high-content/high-throughput imaging methods that enable tracking the bacillus inside host settings, taking into account the heterogeneity of colonization. PMID:25779330

  7. Mechanism of ESAT-6 membrane interaction and its roles in pathogenesis of Mycobacterium tuberculosis.

    PubMed

    Peng, Xiuli; Sun, Jianjun

    2016-06-15

    The 6-kDa early secreted antigenic target (ESAT-6; EsxA) of Mycobacterium tuberculosis was first identified as a potent T-cell antigen, and it is now recognized as a pore-forming toxin that is essential for virulence of M. tuberculosis. ESAT-6 is secreted through the ESX-1 secretion system (Type VII) of M. tuberculosis and has been implicated to mediate mycobacterial cytosolic translocation within the host macrophages by rupturing the phagosomal membranes. Recent studies have made significant progresses in understanding of the mechanism of ESAT-6 membrane interaction and its role in M. tuberculosis pathogenesis, but important questions still remain to be answered. Here, we summarize the current progress in study of ESAT-6 membrane interaction and its roles in pathogenesis and discuss some of the key remaining questions for future investigation. PMID:26456678

  8. Assessment of the serodiagnostic potential of nine novel proteins from Mycobacterium tuberculosis.

    PubMed

    Moran, A J; Treit, J D; Whitney, J L; Abomoelak, B; Houghton, R; Skeiky, Y A; Sampaio, D P; Badaró, R; Nano, F E

    2001-04-20

    To identify antigens that would improve the accuracy of serological diagnosis of active tuberculosis, we cloned the genes encoding nine potentially immunogenic secreted or surface-associated proteins of Mycobacterium tuberculosis. Recombinant proteins were reacted with sera from HIV-negative individuals with extrapulmonary tuberculosis (EP-TB) or HIV-positive individuals with pulmonary tuberculosis (TBH). Specific and high level antibody responses were obtained for four recombinant proteins, of which antigen GST-822 was recognized by 60% of EP-TB and 42% of TBH and antigen MBP-506 was recognized by 45% of EP-TB and 61% of TBH. These results suggest that these proteins are strong candidates as subunits in a polyvalent serodiagnostic test. PMID:11325550

  9. Molecular epidemiology of Mycobacterium tuberculosis in the United States-Affiliated Pacific Islands.

    PubMed

    Bamrah, Sapna; Desmond, Edward; Ghosh, Smita; France, Anne Marie; Kammerer, J Steve; Cowan, Lauren S; Heetderks, Andrew; Forbes, Alstead; Moonan, Patrick K

    2014-01-01

    The United States-Affiliated Pacific Islands (USAPI) are part of the US National Tuberculosis (TB) Surveillance System and use laboratory services contracted through a cooperative agreement with the Centers for Disease Control and Prevention (CDC). In 2004, the CDC established the National Tuberculosis Genotyping Service, a system to genotype 1 isolate from each culture-confirmed case of TB. To describe the molecular epidemiology of TB in the region, we examined all Mycobacterium tuberculosis isolates submitted for genotyping from January 1, 2004, to December 31, 2008. Over this time period, the USAPI jurisdictions reported 1339 verified TB cases to the National Tuberculosis Surveillance System. Among 419 (31%) reported culture-confirmed TB cases, 352 (84%) had complete genotype results. Routine TB genotyping allowed, for the first time, an exploration of the molecular epidemiology of TB in the USAPI. PMID:23239749

  10. Comparative Study of Activities of a Diverse Set of Antimycobacterial Agents against Mycobacterium tuberculosis and Mycobacterium ulcerans.

    PubMed

    Scherr, Nicole; Pluschke, Gerd; Panda, Manoranjan

    2016-05-01

    A library of compounds covering a broad chemical space was selected from a tuberculosis drug development program and was screened in a whole-cell assay against Mycobacterium ulcerans, the causative agent of the necrotizing skin disease Buruli ulcer. While a number of potent antitubercular agents were only weakly active or inactive against M. ulcerans, five compounds showed high activity (90% inhibitory concentration [IC90], ≤1 μM), making screening of focused antitubercular libraries a good starting point for lead generation against M. ulcerans. PMID:26883701

  11. The Influence of Host and Bacterial Genotype on the Development of Disseminated Disease with Mycobacterium tuberculosis

    PubMed Central

    Caws, Maxine; Thwaites, Guy; Dunstan, Sarah; Hawn, Thomas R.; Thi Ngoc Lan, Nguyen; Thuong, Nguyen Thuy Thuong; Stepniewska, Kasia; Huyen, Mai Nguyet Thu; Bang, Nguyen Duc; Huu Loc, Tran; Gagneux, Sebastien; van Soolingen, Dick; Kremer, Kristin; van der Sande, Marianne; Small, Peter; Thi Hoang Anh, Phan; Chinh, Nguyen Tran; Thi Quy, Hoang; Thi Hong Duyen, Nguyen; Quang Tho, Dau; Hieu, Nguyen T.; Torok, Estee; Hien, Tran Tinh; Dung, Nguyen Huy; Thi Quynh Nhu, Nguyen; Duy, Phan Minh; van Vinh Chau, Nguyen; Farrar, Jeremy

    2008-01-01

    The factors that govern the development of tuberculosis disease are incompletely understood. We hypothesized that some strains of Mycobacterium tuberculosis (M. tuberculosis) are more capable of causing disseminated disease than others and may be associated with polymorphisms in host genes responsible for the innate immune response to infection. We compared the host and bacterial genotype in 187 Vietnamese adults with tuberculous meningitis (TBM) and 237 Vietnamese adults with uncomplicated pulmonary tuberculosis. The host genotype of tuberculosis cases was also compared with the genotype of 392 cord blood controls from the same population. Isolates of M. tuberculosis were genotyped by large sequence polymorphisms. The hosts were defined by polymorphisms in genes encoding Toll-interleukin 1 receptor domain containing adaptor protein (TIRAP) and Toll-like receptor-2 (TLR-2). We found a significant protective association between the Euro-American lineage of M. tuberculosis and pulmonary rather than meningeal tuberculosis (Odds ratio (OR) for causing TBM 0.395, 95% confidence intervals (C.I.) 0.193–0.806, P = 0.009), suggesting these strains are less capable of extra-pulmonary dissemination than others in the study population. We also found that individuals with the C allele of TLR-2 T597C allele were more likely to have tuberculosis caused by the East-Asian/Beijing genotype (OR = 1.57 [95% C.I. 1.15–2.15]) than other individuals. The study provides evidence that M. tuberculosis genotype influences clinical disease phenotype and demonstrates, for the first time, a significant interaction between host and bacterial genotypes and the development of tuberculosis. PMID:18369480

  12. Multiscale Model of Mycobacterium tuberculosis Infection Maps Metabolite and Gene Perturbations to Granuloma Sterilization Predictions.

    PubMed

    Pienaar, Elsje; Matern, William M; Linderman, Jennifer J; Bader, Joel S; Kirschner, Denise E

    2016-05-01

    Granulomas are a hallmark of tuberculosis. Inside granulomas, the pathogen Mycobacterium tuberculosis may enter a metabolically inactive state that is less susceptible to antibiotics. Understanding M. tuberculosis metabolism within granulomas could contribute to reducing the lengthy treatment required for tuberculosis and provide additional targets for new drugs. Two key adaptations of M. tuberculosis are a nonreplicating phenotype and accumulation of lipid inclusions in response to hypoxic conditions. To explore how these adaptations influence granuloma-scale outcomes in vivo, we present a multiscale in silico model of granuloma formation in tuberculosis. The model comprises host immunity, M. tuberculosis metabolism, M. tuberculosis growth adaptation to hypoxia, and nutrient diffusion. We calibrated our model to in vivo data from nonhuman primates and rabbits and apply the model to predict M. tuberculosis population dynamics and heterogeneity within granulomas. We found that bacterial populations are highly dynamic throughout infection in response to changing oxygen levels and host immunity pressures. Our results indicate that a nonreplicating phenotype, but not lipid inclusion formation, is important for long-term M. tuberculosis survival in granulomas. We used virtual M. tuberculosis knockouts to predict the impact of both metabolic enzyme inhibitors and metabolic pathways exploited to overcome inhibition. Results indicate that knockouts whose growth rates are below ∼66% of the wild-type growth rate in a culture medium featuring lipid as the only carbon source are unable to sustain infections in granulomas. By mapping metabolite- and gene-scale perturbations to granuloma-scale outcomes and predicting mechanisms of sterilization, our method provides a powerful tool for hypothesis testing and guiding experimental searches for novel antituberculosis interventions. PMID:26975995

  13. The DosR Regulon Modulates Adaptive Immunity and Is Essential for Mycobacterium tuberculosis Persistence

    PubMed Central

    Mehra, Smriti; Foreman, Taylor W.; Didier, Peter J.; Ahsan, Muhammad H.; Hudock, Teresa A.; Kissee, Ryan; Golden, Nadia A.; Gautam, Uma S.; Johnson, Ann-Marie; Alvarez, Xavier; Russell-Lodrigue, Kasi E.; Doyle, Lara A.; Roy, Chad J.; Niu, Tianhua; Blanchard, James L.; Khader, Shabaana A.; Lackner, Andrew A.; Sherman, David R.

    2015-01-01

    Rationale: Hypoxia promotes dormancy by causing physiologic changes to actively replicating Mycobacterium tuberculosis. DosR controls the response of M. tuberculosis to hypoxia. Objectives: To understand DosR's contribution in the persistence of M. tuberculosis, we compared the phenotype of various DosR regulon mutants and a complemented strain to M. tuberculosis in macaques, which faithfully model M. tuberculosis infection. Methods: We measured clinical and microbiologic correlates of infection with M. tuberculosis relative to mutant/complemented strains in the DosR regulon, studied lung pathology and hypoxia, and compared immune responses in lung using transcriptomics and flow cytometry. Measurements and Main Results: Despite being able to replicate initially, mutants in DosR regulon failed to persist or cause disease. On the contrary, M. tuberculosis and a complemented strain were able to establish infection and tuberculosis. The attenuation of pathogenesis in animals infected with the mutants coincided with the appearance of a Th1 response and organization of hypoxic lesions wherein M. tuberculosis expressed dosR. The lungs of animals infected with the mutants (but not the complemented strain) exhibited early transcriptional signatures of T-cell recruitment, activation, and proliferation associated with an increase of T cells expressing homing and proliferation markers. Conclusions: Delayed adaptive responses, a hallmark of M. tuberculosis infection, not only lead to persistence but also interfere with the development of effective antituberculosis vaccines. The DosR regulon therefore modulates both the magnitude and the timing of adaptive immune responses in response to hypoxia in vivo, resulting in persistent infection. Hence, DosR regulates key aspects of the M. tuberculosis life cycle and limits lung pathology. PMID:25730547

  14. Analysis of genetic polymorphism in the phospholipase region of Mycobacterium tuberculosis.

    PubMed Central

    Vera-Cabrera, L; Howard, S T; Laszlo, A; Johnson, W M

    1997-01-01

    mtp40 was originally identified as a short genomic region that was found in strains of Mycobacterium tuberculosis but not in Mycobacterium bovis. Subsequent studies have revealed that the sequence is part of the mpcA gene, which encodes a phospholipase C. To investigate further the distribution of the mtp40 sequence, we analyzed strains of the M. tuberculosis complex by PCR and were able to amplify the mtp40 sequence in 90 of 94 strains of M. tuberculosis and in 2 strains of Mycobacterium microti but not in M. bovis or M. bovis BCG. Based on this, we developed a dot blot assay using genomic DNA which allows M. bovis to be distinguished from the majority of M. tuberculosis strains. We also probed Southern blots of 140 clinical isolates of M. tuberculosis to determine the frequency of strains lacking mtp40. This revealed an unexpected polymorphism in the phospholipase region. Two fragments were detected in 57% of samples. The expected fragment of 0.75 kbp corresponds to the region of mpcA containing mtp40. A 2.1-kbp fragment was observed to belong to a recently discovered second phospholipase gene, mpcB. In addition, some strains appeared to lack both genes, while others showed only the presence of mpcA. A few strains had additional bands, suggesting the existence of other homologs to the two phospholipase genes. We also detected the insertion of IS6110 in the mpcA coding region of one strain. The absence of these genes in some clinical isolates raises questions about their function during infection and in the development of tuberculosis disease in humans. PMID:9114405

  15. Mycobacterium tuberculosis infection in a canary (Serinus canana L.) and a blue-fronted Amazon parrot (Amazona amazona aestiva).

    PubMed

    Hoop, Richard K

    2002-01-01

    I report two cases of mycobacteriosis in pet birds due to Mycobacterium tuberculosis and discuss the zoonotic implications. The canary with a tuberculous knot in the lung is the first description of M. tuberculosis in a nonpsittacine bird species. PMID:12061666

  16. Use of GeneXpert Mycobacterium tuberculosis/rifampicin for rapid detection of rifampicin resistant Mycobacterium tuberculosis strains of clinically suspected multi-drug resistance tuberculosis cases

    PubMed Central

    Guenaoui, Kheira; Ouardi, Aissa; Zeggai, Soumia; Sellam, Feriel; Bekri, Farid; Cherif Touil, Sakina

    2016-01-01

    Background Multi-drug resistance (MDR) TB is defined as tuberculosis (TB) disease caused by a strain of Mycobacterium tuberculosis (MTB) that was resistant to at least isoniazid and rifampicin (RIF). Emerging Multidrug-Resistant TB is one of the major concerns of health policy and rapid detection of M. tuberculosis and detection of RIF resistance in infected patients are essential for disease management. The aim of this study was to evaluate patterns of RIF resistance in cases of sputum positive pulmonary TB by using GeneXpert MTB/RIF and comparing between phenotypic and genotypic testing of RIF resistance in MTB strains of clinically suspected MDR-TB isolated cases in western Algeria. Methods In this study 50 sputum positive cases of pulmonary TB who were potential MDR suspect were included. Their sputum samples were collected and subjected to sputum smear microscopy, culture and conventional MTB/RIF test followed by GeneXpert MTB/RIF assay. Results Of total 50 cases included in this study, MTB was detected in all patients (100%) by GeneXpert MTB/RIF. However, RIF’s resistance was detected in only 21 cases (42%) by GeneXpert MTB/RIF. All RIF resistant strains detected by GeneXpert MTB/RIF were phenotypically confirmed as MDR strains. 42.85% of cases were retreatment failure cases, retreatment cases smear positive at 4 months were 23.82%. While 19.05% of cases were retreatment cases smear positive at diagnosis, and 14.28% patient had history of contact with MDR-TB. Sensitivity, specificity, positive predictive value and negative predictive value of Xpert MTB/RIF to detect RIF resistance in comparison to conventional phenotypic drug susceptibility technique were found equal to the rates of 100%, 100%, 100% and 100%, respectively. Conclusions GeneXpert MTB/RIF assay is efficient and reliable technique for the rapid diagnostic of TB. It’s simplicity, high sensitivity and specificity for RIF resistance detection make this technique a very attractive tool for

  17. New drug susceptibility test for Mycobacterium tuberculosis using the hybridization protection assay.

    PubMed Central

    Miyamoto, J; Koga, H; Kohno, S; Tashiro, T; Hara, K

    1996-01-01

    We developed a novel method for early detection of drug-resistant strains of Mycobacterium tuberculosis by using the hybridization protection assay (HPA). The number of viable bacteria during the incubation period correlated well with the number of relative light units measured by the HPA. In addition, the relative light unit values of susceptible strains on the first, third and fifth days of incubation were significantly different from those of resistant strains for both isoniazid and rifampin. Our results suggest that after isolation of the organism from clinical specimens, drug-resistant strains of M. tuberculosis are accurately detected by the HPA even after 1 day of incubation with the drug. PMID:8727932

  18. Molecular epidemiology of Mycobacterium tuberculosis strains isolated from different regions of Italy and Pakistan.

    PubMed Central

    Sechi, L A; Zanetti, S; Delogu, G; Montinaro, B; Sanna, A; Fadda, G

    1996-01-01

    The use of the (GTG)5 oligonucleotide, a repetitive marker in the Mycobacterium tuberculosis chromosome, as a primer in association with an IS6110 outlooking primer has been successfully applied to a PCR-based fingerprinting method. This method classified 62 strains of M. tuberculosis, isolated from human immunodeficiency virus-seropositive and -seronegative patients in different regions of Italy and Pakistan, as having 53 different patterns. The results were compared with traditional IS6110 fingerprinting, by which 47 distinct patterns were observed. PMID:8784602

  19. Genome sequencing and annotation of multidrug resistant Mycobacterium tuberculosis (MDR-TB) PR10 strain

    PubMed Central

    Halim, Mohd Zakihalani A.; Jaafar, Mohammad Maaruf; Teh, Lay Kek; Ismail, Mohamad Izwan; Lee, Lian Shien; Ngeow, Yun Fong; Nor, Norazmi Mohd; Zainuddin, Zainul Fadziruddin; Tang, Thean Hock; Najimudin, Mohd Nazalan Mohd; Salleh, Mohd Zaki

    2016-01-01

    Here, we report the draft genome sequence and annotation of a multidrug resistant Mycobacterium tuberculosis strain PR10 (MDR-TB PR10) isolated from a patient diagnosed with tuberculosis. The size of the draft genome MDR-TB PR10 is 4.34 Mbp with 65.6% of G + C content and consists of 4637 predicted genes. The determinants were categorized by RAST into 400 subsystems with 4286 coding sequences and 50 RNAs. The whole genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession number CP010968. PMID:26981419

  20. The MTCY428.08 Gene of Mycobacterium tuberculosis Codes for NAD+ Synthetase

    PubMed Central

    Cantoni, Rita; Branzoni, Manuela; Labò, Monica; Rizzi, Menico; Riccardi, Giovanna

    1998-01-01

    The product of the MTCY428.08 gene of Mycobacterium tuberculosis shows sequence homology with several NAD+ synthetases. The MTCY428.08 gene was cloned into the expression vectors pGEX-4T-1 and pET-15b. Expression in Escherichia coli led to overproduction of glutathione S-transferase fused and His6-tagged gene products, which were enzymatically assayed for NAD synthetase activity. Our results demonstrate that the MTCY428.08 gene of M. tuberculosis is the structural gene for NAD+ synthetase. PMID:9620974

  1. Genome sequencing and annotation of multidrug resistant Mycobacterium tuberculosis (MDR-TB) PR10 strain.

    PubMed

    Halim, Mohd Zakihalani A; Jaafar, Mohammad Maaruf; Teh, Lay Kek; Ismail, Mohamad Izwan; Lee, Lian Shien; Ngeow, Yun Fong; Nor, Norazmi Mohd; Zainuddin, Zainul Fadziruddin; Tang, Thean Hock; Najimudin, Mohd Nazalan Mohd; Salleh, Mohd Zaki

    2016-03-01

    Here, we report the draft genome sequence and annotation of a multidrug resistant Mycobacterium tuberculosis strain PR10 (MDR-TB PR10) isolated from a patient diagnosed with tuberculosis. The size of the draft genome MDR-TB PR10 is 4.34 Mbp with 65.6% of G + C content and consists of 4637 predicted genes. The determinants were categorized by RAST into 400 subsystems with 4286 coding sequences and 50 RNAs. The whole genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession number CP010968. PMID:26981419

  2. [New technologies in the determination of drug susceptibility in Mycobacterium tuberculosis].

    PubMed

    Skotnikova, O I; Mikhaĭlovich, V M; Nosova, E Iu; Lapa, S A; Griadunov, D A; Donnikov, M Iu; Badleeva, M V; Galkina, K Iu; Dorozhkova, I R; Litvinov, V I; Zasedatelev, A S; Moroz, A M; Mirzabekov, A D

    2004-01-01

    A variety of mutations in the genes rpoB, katG, inhA, ahpC, kasA was studied by using different molecular biological methods (conformational polymorphism of single-chain fragments, heteroduplex analysis, biochips) in rifampicin- and isoniazid-resistant Mycobacterium tuberculosis (MBT) strains isolated from patients with pulmonary tuberculosis. Twenty-nine mutation combinations were identified in the MBT strains. The use of biochips is the most promising method for identifying the type of mutations responsible for the simultaneous resistance to rifampicin and isoniazid. Detection of several MBT strains in one patient requires the use a combination of molecular biological and microbiological studies. PMID:15315132

  3. Novel mutations in ndh in isoniazid-resistant Mycobacterium tuberculosis isolates.

    PubMed

    Lee, A S; Teo, A S; Wong, S Y

    2001-07-01

    Novel mutations in NADH dehydrogenase (ndh) were detected in 8 of 84 (9.5%) isoniazid (INH)-resistant isolates (T110A [n = 1], R268H [n = 7]), but not in 22 INH-susceptible isolates of Mycobacterium tuberculosis. Significantly, all eight isolates with mutations at ndh did not have mutations at katG, kasA, or the promoter regions of inhA or ahpC, except for one isolate. Mutations in ndh appear to be an additional molecular mechanism for isoniazid resistance in M. tuberculosis. PMID:11408244

  4. Purification, crystallization and preliminary X-ray crystallographic studies of Rv3705c from Mycobacterium tuberculosis

    SciTech Connect

    Lu, Feifei; Gao, Feng; Li, Honglin; Gong, Weimin; Zhou, Lin; Bi, Lijun

    2014-07-23

    The cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of Rv3705c from M. tuberculosis are described. The conserved protein Rv3705c from Mycobacterium tuberculosis has been cloned, expressed, purified and crystallized by the sitting-drop vapour-diffusion method using PEG 3350 as a precipitant. The Rv3705c crystals exhibited space group P6{sub 1}22 or P6{sub 5}22, with unit-cell parameters a = b = 198.0, c = 364.1 Å, α = β = 90, γ = 120°, and diffracted to a resolution of 3.3 Å.

  5. Multicenter evaluation of the nitrate reductase assay for drug resistance detection of Mycobacterium tuberculosis.

    PubMed

    Martin, Anandi; Montoro, Ernesto; Lemus, Dihadenys; Simboli, Norberto; Morcillo, Nora; Velasco, Maritza; Chauca, José; Barrera, Lucía; Ritacco, Viviana; Portaels, Françoise; Palomino, Juan Carlos

    2005-11-01

    The performance of the nitrate reductase assay was evaluated in a multicenter laboratory study to detect resistance of Mycobacterium tuberculosis to the first-line anti-tuberculosis drugs rifampicin, isoniazid, ethambutol and streptomycin using a set of coded isolates. Compared with the gold standard proportion method on Löwenstein-Jensen medium, the assay was highly accurate in detecting resistance to rifampicin, isoniazid and ethambutol with an accuracy of 98%, 96.6% and 97.9%, respectively. For streptomycin, discrepant results were obtained with an overall accuracy of 85.3%. The assay proved easy to be implemented in countries with limited laboratory facilities. PMID:15893391

  6. Mycobacterium tuberculosis Lipolytic Enzymes as Potential Biomarkers for the Diagnosis of Active Tuberculosis

    PubMed Central

    Brust, Belinda; Lecoufle, Mélanie; Tuaillon, Edouard; Dedieu, Luc; Canaan, Stéphane; Valverde, Viviane; Kremer, Laurent

    2011-01-01

    Background New diagnosis tests are urgently needed to address the global tuberculosis (TB) burden and to improve control programs especially in resource-limited settings. An effective in vitro diagnostic of TB based on serological methods would be regarded as an attractive progress because immunoassays are simple, rapid, inexpensive, and may offer the possibility to detect cases missed by standard sputum smear microscopy. However, currently available serology tests for TB are highly variable in sensitivity and specificity. Lipolytic enzymes have recently emerged as key factors in lipid metabolization during dormancy and/or exit of the non-replicating growth phase, a prerequisite step of TB reactivation. The focus of this study was to analyze and compare the potential of four Mycobacterium tuberculosis lipolytic enzymes (LipY, Rv0183, Rv1984c and Rv3452) as new markers in the serodiagnosis of active TB. Methods Recombinant proteins were produced and used in optimized ELISA aimed to detect IgG and IgM serum antibodies against the four lipolytic enzymes. The capacity of the assays to identify infection was evaluated in patients with either active TB or latent TB and compared with two distinct control groups consisting of BCG-vaccinated blood donors and hospitalized non-TB individuals. Results A robust humoral response was detected in patients with active TB whereas antibodies against lipolytic enzymes were infrequently detected in either uninfected groups or in subjects with latent infection. High specifity levels, ranging from 93.9% to 97.5%, were obtained for all four antigens with sensitivity values ranging from 73.4% to 90.5%, with Rv3452 displaying the highest performances. Patients with active TB usually exhibited strong IgG responses but poor IgM responses. Conclusion These results clearly indicate that the lipolytic enzymes tested are strongly immunogenic allowing to distinguish active from latent TB infections. They appear as potent biomarkers providing high

  7. Exposure to Mycobacterium tuberculosis during Flexible Bronchoscopy in Patients with Unexpected Pulmonary Tuberculosis

    PubMed Central

    Na, Hae Jung; Eom, Jung Seop; Lee, Geewon; Mok, Jeong Ha; Kim, Mi Hyun; Lee, Kwangha; Kim, Ki Uk; Lee, Min Ki

    2016-01-01

    Objective Recent guidelines recommend the use by healthcare personnel of a fit-tested N95 particulate respirator or higher-grade respiratory precaution in a patient undergoing bronchoscopy when pulmonary tuberculosis (PTB) is suspected. However, PTB may be unexpectedly diagnosed in this setting and therefore not evaluated, resulting in the unexpected exposure to Mycobacterium tuberculosis (MTB) of healthcare workers in the bronchoscopy suite. Here, we examined the incidence of unexpected exposure to MTB during flexible bronchoscopy and determined the exposure-related factors. Methods Between 2011 and 2013, a retrospective study was conducted to evaluate unexpected diagnoses of PTB in the bronchoscopy suite. During the study period, 1650 consecutive patients for whom previous CT scans were available and who underwent bronchoscopy for respiratory disease other than PTB were included. The results of bronchial washing, bronchoalveolar lavage, and post-bronchoscopic sputum were reviewed. Results PTB was unexpectedly diagnosed in 76 patients (4.6%). The presence of anthracofibrosis [odds ratio (OR), 3.878; 95% confidence interval (CI), 1.291–11.650; P = 0.016), bronchiectasis (OR, 1.974; 95% CI, 1.095–3.557; P = 0.024), or atelectasis (OR, 1.740; 95% CI, 1.010–2.903; P = 0.046) as seen on chest CT scan was independently associated with unexpected PTB. Patients with both anthracofibrosis and atelectasis were at much higher risk of unexpected PTB (OR, 4.606; 95% CI, 1.383–15.342; P = 0.013). Conclusions The risk of MTB exposure by healthcare personnel in the bronchoscopy suite due to patients with undiagnosed PTB has been underestimated. Therefore, in geographic regions with an intermediate PTB prevalence, such as South Korea (97/100,000 persons per year), higher-grade respiratory precaution, such as a fit-tested N95 particulate respirator, should be considered to prevent occupational exposure to MTB during routine bronchoscopy, especially in patients with CT

  8. Comprehensive multicenter evaluation of a new line probe assay kit for identification of Mycobacterium species and detection of drug-resistant Mycobacterium tuberculosis.

    PubMed

    Mitarai, Satoshi; Kato, Seiya; Ogata, Hideo; Aono, Akio; Chikamatsu, Kinuyo; Mizuno, Kazue; Toyota, Emiko; Sejimo, Akiko; Suzuki, Katsuhiro; Yoshida, Shiomi; Saito, Takefumi; Moriya, Ataru; Fujita, Akira; Sato, Shuko; Matsumoto, Tomoshige; Ano, Hiromi; Suetake, Toshinori; Kondo, Yuji; Kirikae, Teruo; Mori, Toru

    2012-03-01

    We evaluated a new line probe assay (LiPA) kit to identify Mycobacterium species and to detect mutations related to drug resistance in Mycobacterium tuberculosis. A total of 554 clinical isolates of Mycobacterium tuberculosis (n = 316), Mycobacterium avium (n = 71), Mycobacterium intracellulare (n = 51), Mycobacterium kansasii (n = 54), and other Mycobacterium species (n = 62) were tested with the LiPA kit in six hospitals. The LiPA kit was also used to directly test 163 sputum specimens. The results of LiPA identification of Mycobacterium species in clinical isolates were almost identical to those of conventional methods. Compared with standard drug susceptibility testing results for the clinical isolates, LiPA showed a sensitivity and specificity of 98.9% and 97.3%, respectively, for detecting rifampin (RIF)-resistant clinical isolates; 90.6% and 100%, respectively, for isoniazid (INH) resistance; 89.7% and 96.0%, respectively, for pyrazinamide (PZA) resistance; and 93.0% and 100%, respectively, for levofloxacin (LVX) resistance. The LiPA kit could detect target species directly in sputum specimens, with a sensitivity of 85.6%. Its sensitivity and specificity for detecting RIF-, PZA-, and LVX-resistant isolates in the sputum specimens were both 100%, and those for detecting INH-resistant isolates were 75.0% and 92.9%, respectively. The kit was able to identify mycobacterial bacilli at the species level, as well as drug-resistant phenotypes, with a high sensitivity and specificity. PMID:22205814

  9. A convenient synthesis and screening of benzosuberone bearing 1,2,3-triazoles against Mycobacterium tuberculosis.

    PubMed

    Sajja, Yasodakrishna; Vanguru, Sowmya; Jilla, Lavanya; Vulupala, Hanmanth Reddy; Bantu, Rajashaker; Yogeswari, Perumal; Sriram, Dharmarajan; Nagarapu, Lingaiah

    2016-09-01

    A series of benzosuberone bearing 1,2,3-triazoles were rationally designed and alkyl/aryl groups appended on 1,2,3-triazole derivatives 5a-o were synthesized using click chemistry and evaluated for their in vitro antimycobacterial activity against Mycobacterium tuberculosis H37Rv (ATCC27294). Compounds 5h (MIC: 3.125μg/mL) and 5l, 5m, 5o (MIC: 6.25μg/mL) exhibited promising hits. This is the first Letter on the synthesis and in vitro antimycobacterial activity against Mycobacterium tuberculosis H37Rv of benzosuberone alkyl/aryl groups appended on 1,2,3-triazole derivatives. PMID:27476139

  10. Predominance of modern Mycobacterium tuberculosis strains and active transmission of Beijing sublineage in Jayapura, Indonesia Papua.

    PubMed

    Chaidir, Lidya; Sengstake, Sarah; de Beer, Jessica; Oktavian, Antonius; Krismawati, Hana; Muhapril, Erfin; Kusumadewi, Inri; Annisa, Jessi; Anthony, Richard; van Soolingen, Dick; Achmad, Tri Hanggono; Marzuki, Sangkot; Alisjahbana, Bachti; van Crevel, Reinout

    2016-04-01

    Mycobacterium tuberculosis genotype distribution is different between West and Central Indonesia, but there are no data on the most Eastern part, Papua. We aimed to identify the predominant genotypes of M. tuberculosis responsible for tuberculosis in coastal Papua, their transmission, and the association with patient characteristics. A total of 199 M. tuberculosis isolates were collected. Spoligotyping was applied to describe the population structure of M. tuberculosis, lineage identification was performed using a combination of lineage-specific markers, and genotypic clusters were identified using a combination of 24-locus-MIRU-VNTR and spoligotyping. A high degree of genetic diversity was observed among isolates based on their spoligopatterns. Strains from modern lineage 4 made up almost half of strains (46.9%), being more abundant than the ancient lineage 1 (33.7%), and modern lineage 2 (19.4%). Thirty-five percent of strains belonged to genotypic clusters, especially strains in the Beijing genotype. Previous TB treatment and mutations associated with drug resistance were more common in patients infected with strains of the Beijing genotype. Papua shows a different distribution of M. tuberculosis genotypes compared to other parts of Indonesia. Clustering and drug resistance of modern strains recently introduced to Papua may contribute to the high tuberculosis burden in this region. PMID:26825253

  11. Bim is a crucial regulator of apoptosis induced by Mycobacterium tuberculosis

    PubMed Central

    Aguiló, N; Uranga, S; Marinova, D; Martín, C; Pardo, J

    2014-01-01

    Mycobacterium tuberculosis, the causative agent of tuberculosis, induces apoptosis in infected macrophages in vitro and in vivo. However, the molecular mechanism controlling this process is not known. In order to study the involvement of the mitochondrial apoptotic pathway in M. tuberculosis-induced apoptosis, we analysed cell death in M. tuberculosis-infected embryonic fibroblasts (MEFs) derived from different knockout mice for genes involved in this route. We found that apoptosis induced by M. tuberculosis is abrogated in the absence of Bak and Bax, caspase 9 or the executioner caspases 3 and 7. Notably, we show that MEF deficient in the BH3-only BCL-2-interacting mediator of cell death (Bim) protein were also resistant to this process. The relevance of these results has been confirmed in the mouse macrophage cell line J774, where cell transfection with siRNA targeting Bim impaired apoptosis induced by virulent mycobacteria. Notably, only infection with a virulent strain, but not with attenuated ESX-1-defective strains, such as Bacillus Calmette-Guerin and live-attenuated M. tuberculosis vaccine strain MTBVAC, induced Bim upregulation and apoptosis, probably implicating virulence factor early secreted antigenic target 6-kDa protein in this process. Our results suggest that Bim upregulation and apoptosis is mediated by the p38MAPK-dependent pathway. Our findings show that Bim is a master regulator of apoptosis induced by M. tuberculosis. PMID:25032866

  12. The Cyclic Peptide Ecumicin Targeting ClpC1 Is Active against Mycobacterium tuberculosis In Vivo

    PubMed Central

    Gao, Wei; Kim, Jin-Yong; Anderson, Jeffrey R.; Akopian, Tatos; Hong, Seungpyo; Jin, Ying-Yu; Kandror, Olga; Kim, Jong-Woo; Lee, In-Ae; Lee, Sun-Young; McAlpine, James B.; Mulugeta, Surafel; Sunoqrot, Suhair; Wang, Yuehong; Yang, Seung-Hwan; Yoon, Tae-Mi; Goldberg, Alfred L.; Pauli, Guido F.; Cho, Sanghyun

    2014-01-01

    Drug-resistant tuberculosis (TB) has lent urgency to finding new drug leads with novel modes of action. A high-throughput screening campaign of >65,000 actinomycete extracts for inhibition of Mycobacterium tuberculosis viability identified ecumicin, a macrocyclic tridecapeptide that exerts potent, selective bactericidal activity against M. tuberculosis in vitro, including nonreplicating cells. Ecumicin retains activity against isolated multiple-drug-resistant (MDR) and extensively drug-resistant (XDR) strains of M. tuberculosis. The subcutaneous administration to mice of ecumicin in a micellar formulation at 20 mg/kg body weight resulted in plasma and lung exposures exceeding the MIC. Complete inhibition of M. tuberculosis growth in the lungs of mice was achieved following 12 doses at 20 or 32 mg/kg. Genome mining of lab-generated, spontaneous ecumicin-resistant M. tuberculosis strains identified the ClpC1 ATPase complex as the putative target, and this was confirmed by a drug affinity response test. ClpC1 functions in protein breakdown with the ClpP1P2 protease complex. Ecumicin markedly enhanced the ATPase activity of wild-type (WT) ClpC1 but prevented activation of proteolysis by ClpC1. Less stimulation was observed with ClpC1 from ecumicin-resistant mutants. Thus, ClpC1 is a valid drug target against M. tuberculosis, and ecumicin may serve as a lead compound for anti-TB drug development. PMID:25421483

  13. Single-Cell Elemental Analysis of Bacteria: Quantitative Analysis of Polyphosphates in Mycobacterium tuberculosis

    PubMed Central

    Ward, Sarah K.; Heintz, Joseph A.; Albrecht, Ralph M.; Talaat, Adel M.

    2012-01-01

    More than 1.8 million people die annually from infection with Mycobacterium tuberculosis, the causative agent of tuberculosis. The ability of M. tuberculosis to obtain and distribute micronutrients, including biometals, is known to play a role in its intracellular survival and virulence within a host. Techniques to detect elemental distributions within M. tuberculosis cells have previously been limited to bulk detection methods or low-resolution analyses. Here, we present a method for determining the elemental distribution within M. tuberculosis on a single-cell level, at high (individual nanometer) resolution, using scanning transmission electron microscopy (STEM) in concert with energy-dispersive X-ray spectroscopy (EDS). Results revealed the presence of large polyphosphate granules in all strains of Mycobacteria tested. These persisted even through starvation conditions, and might play a role connected to elemental homeostasis in M. tuberculosis. Associated with the polyphosphate granules were micronutrients such as calcium and magnesium. In addition, we expanded the technique beyond Mycobacteria to show that STEM and EDS could be used as a simple screen to detect the presence or absence of concentrated elements on a single-cell level within all six other bacterial types tested, with minimal processing to the bacteria. Overall, we believe that this technique represents a first step in developing a better understanding of the role that components of the intracellular milieu, including polyphosphates and biometals, play in the pathogenesis of M. tuberculosis, with potential future applications for in vivo analysis. PMID:22919654

  14. cor, a Novel Carbon Monoxide Resistance Gene, Is Essential for Mycobacterium tuberculosis Pathogenesis

    PubMed Central

    Zacharia, Vineetha M.; Manzanillo, Paolo S.; Nair, Vidhya R.; Marciano, Denise K.; Kinch, Lisa N.; Grishin, Nick V.; Cox, Jeffery S.; Shiloh, Michael U.

    2013-01-01

    ABSTRACT Tuberculosis, caused by Mycobacterium tuberculosis, remains a devastating human infectious disease, causing two million deaths annually. We previously demonstrated that M. tuberculosis induces an enzyme, heme oxygenase (HO1), that produces carbon monoxide (CO) gas and that M. tuberculosis adapts its transcriptome during CO exposure. We now demonstrate that M. tuberculosis carries a novel resistance gene to combat CO toxicity. We screened an M. tuberculosis transposon library for CO-susceptible mutants and found that disruption of Rv1829 (carbon monoxide resistance, Cor) leads to marked CO sensitivity. Heterologous expression of Cor in Escherichia coli rescued it from CO toxicity. Importantly, the virulence of the cor mutant is attenuated in a mouse model of tuberculosis. Thus, Cor is necessary and sufficient to protect bacteria from host-derived CO. Taken together, this represents the first report of a role for HO1-derived CO in controlling infection of an intracellular pathogen and the first identification of a CO resistance gene in a pathogenic organism. PMID:24255121

  15. CsoR Is Essential for Maintaining Copper Homeostasis in Mycobacterium tuberculosis

    PubMed Central

    Marcus, Sarah A.; Sidiropoulos, Sarah W.; Steinberg, Howard; Talaat, Adel M.

    2016-01-01

    Mycobacterium tuberculosis, a pathogen infecting one third of the world population, faces numerous challenges within the host, including high levels of copper. We have previously shown that M. tuberculosis CsoR is a copper inducible transcriptional regulator. Here we examined the hypothesis that csoR is necessary for maintaining copper homeostasis and surviving under various stress conditions. With an unmarked csoR knockout strain, we were able to characterize the role of csoR in M. tuberculosis as it faced copper and host stress. Growth under high levels of copper demonstrated that M. tuberculosis survives copper stress significantly better in the absence of csoR. Yet under minimal levels of copper, differential expression analysis revealed that the loss of csoR results in a cell wide hypoxia-type stress response with the induction of the DosR regulon. Despite the stress placed on M. tuberculosis by the loss of csoR, survival of the knockout strain was increased compared to wild type during the early chronic stages of mouse infection, suggesting that csoR could play an active role in modulating M. tuberculosis fitness within the host. Overall, analysis of CsoR provided an increased understanding of the M. tuberculosis copper response with implications for other intracellular pathogens harboring CsoR. PMID:26999439

  16. Systems Biology-Based Identification of Mycobacterium tuberculosis Persistence Genes in Mouse Lungs

    PubMed Central

    Dutta, Noton K.; Bandyopadhyay, Nirmalya; Veeramani, Balaji; Lamichhane, Gyanu; Karakousis, Petros C.; Bader, Joel S.

    2014-01-01

    ABSTRACT Identifying Mycobacterium tuberculosis persistence genes is important for developing novel drugs to shorten the duration of tuberculosis (TB) treatment. We developed computational algorithms that predict M. tuberculosis genes required for long-term survival in mouse lungs. As the input, we used high-throughput M. tuberculosis mutant library screen data, mycobacterial global transcriptional profiles in mice and macrophages, and functional interaction networks. We selected 57 unique, genetically defined mutants (18 previously tested and 39 untested) to assess the predictive power of this approach in the murine model of TB infection. We observed a 6-fold enrichment in the predicted set of M. tuberculosis genes required for persistence in mouse lungs relative to randomly selected mutant pools. Our results also allowed us to reclassify several genes as required for M. tuberculosis persistence in vivo. Finally, the new results implicated additional high-priority candidate genes for testing. Experimental validation of computational predictions demonstrates the power of this systems biology approach for elucidating M. tuberculosis persistence genes. PMID:24549847

  17. Evaluation of the upgraded amplified Mycobacterium tuberculosis direct test (gen-probe) for direct detection of Mycobacterium tuberculosis in respiratory and non-respiratory specimens.

    PubMed

    Alcalá, L; Ruiz-Serrano, M J; Hernangómez, S; Marín, M; García de Viedma, D; San Juan, R; Bouza, E

    2001-01-01

    We evaluated the upgraded Amplified Mycobacterium Tuberculosis Direct Test kit (AMTD) (Gen-Probe Inc.) for the direct detection of Mycobacterium tuberculosis in respiratory and non-respiratory specimens, and compared the results between the traditional 30,000 RLUs cutoff criteria (C) and three equivocal ranges (30,000-100,000, R1; 30,000-500,000, R2; and 30,000-1,000,000, R3). We tested 663 respiratory and 238 non-respiratory samples from 464 patients. The gold standard was considered to be the combination of culture and clinical data. One hundred and nineteen samples were from 56 patients with pulmonary tuberculosis, and 36 samples were from 19 patients with extrapulmonary tuberculosis. When C criteria was applied, the sensitivity and specificity values were 90.8 and 93.0% for respiratory specimens, while they were 88.9 and 92.1% for non-respiratory specimens (p = NS). The sensitivity was significantly higher in smear-positive specimens (96.7%) than in smear-negative ones (81.0%) (p < 0.05). When compared with C criteria, the overall sensitivity was maintained at 90.3% for R1 criteria, and slightly decreased to 89.7% for R2 and R3 criteria (p = NS). Overall, specificity increased significantly from 92.9% (C) to 97.5% (R1), 99.1% (R2), and 99.2% (R3). Application of R2 or R3 criteria improved significantly the specificity of the test with little decrease in sensitivity. PMID:11687314

  18. Identification and Genotyping of Mycobacterium tuberculosis Isolated From Water and Soil Samples of a Metropolitan City

    PubMed Central

    Velayati, Ali Akbar; Farnia, Parissa; Mozafari, Mohadese; Malekshahian, Donya; Farahbod, Amir Masoud; Seif, Shima; Rahideh, Snaz

    2015-01-01

    BACKGROUND: The potential role of environmental Mycobacterium tuberculosis in the epidemiology of TB remains unknown. We investigated the transmission of M tuberculosis from humans to the environment and the possible transmission of M tuberculosis from the environment to humans. METHODS: A total of 1,500 samples were collected from three counties of the Tehran, Iran metropolitan area from February 2012 to January 2014. A total of 700 water samples (47%) and 800 soil samples (53%) were collected. Spoligotyping and the mycobacterial interspersed repetitive units-variable number of tandem repeats typing method were performed on DNA extracted from single colonies. Genotypes of M tuberculosis strains isolated from the environment were compared with the genotypes obtained from 55 patients with confirmed pulmonary TB diagnosed during the study period in the same three counties. RESULTS: M tuberculosis was isolated from 11 of 800 soil samples (1%) and 71 of 700 water samples (10%). T family (56 of 82, 68%) followed by Delhi/CAS (11 of 82, 13.4%) were the most frequent M tuberculosis superfamilies in both water and soil samples. Overall, 27.7% of isolates in clusters were related. No related typing patterns were detected between soil, water, and clinical isolates. The most frequent superfamily of M tuberculosis in clinical isolates was Delhi/CAS (142, 30.3%) followed by NEW-1 (127, 27%). The bacilli in contaminated soil (36%) and damp water (8.4%) remained reculturable in some samples up to 9 months. CONCLUSIONS: Although the dominant M tuberculosis superfamilies in soil and water did not correspond to the dominant M tuberculosis family in patients, the presence of circulating genotypes of M tuberculosis in soil and water highlight the risk of transmission. PMID:25340935

  19. Mycobacterium tuberculosis efpA encodes an efflux protein of the QacA transporter family.

    PubMed Central

    Doran, J L; Pang, Y; Mdluli, K E; Moran, A J; Victor, T C; Stokes, R W; Mahenthiralingam, E; Kreiswirth, B N; Butt, J L; Baron, G S; Treit, J D; Kerr, V J; Van Helden, P D; Roberts, M C; Nano, F E

    1997-01-01

    The Mycobacterium tuberculosis H37Rv efpA gene encodes a putative efflux protein, EfpA, of 55,670 Da. The deduced EfpA protein was similar in secondary structure to Pur8, MmrA, TcmA, LfrA, EmrB, and other members of the QacA transporter family (QacA TF) which mediate antibiotic and chemical resistance in bacteria and yeast. The predicted EfpA sequence possessed all transporter motifs characteristic of the QacA TF, including those associated with proton-antiport function and the motif considered to be specific to exporters. The 1,590-bp efpA open reading frame was G+C rich (65%), whereas the 40-bp region immediately upstream had an A+T bias (35% G+C). Reverse transcriptase-PCR assays indicated that efpA was expressed in vitro and in situ. Putative promoter sequences were partially overlapped by the A+T-rich region and by a region capable of forming alternative secondary structures indicative of transcriptional regulation in analogous systems. PCR single-stranded conformational polymorphism analysis demonstrated that these upstream flanking sequences and the 231-bp, 5' coding region are highly conserved among both drug-sensitive and multiply-drug-resistant isolates of M. tuberculosis. The efpA gene was present in the slow-growing human pathogens M. tuberculosis, Mycobacterium leprae, and Mycobacterium bovis and in the opportunistic human pathogens Mycobacterium avium and Mycobacterium intracellular. However, efpA was not present in 17 other opportunistically pathogenic or nonpathogenic mycobacterial species. PMID:9008277

  20. High-contrast imaging of mycobacterium tuberculosis using third-harmonic generation microscopy

    NASA Astrophysics Data System (ADS)

    Kim, Bo Ram; Lee, Eungjang; Park, Seung-Han

    2015-07-01

    Nonlinear optical microcopy has become an important tool in investigating biomaterials due to its various advantages such as label-free imaging capabilities. In particular, it has been shown that third-harmonic generation (THG) signals can be produced at interfaces between an aqueous medium (e.g. cytoplasm, interstitial fluid) and a mineralized lipidic surface. In this work, we have demonstrated that label-free high-contrast THG images of the mycobacterium tuberculosis can be obtained using THG microscopy.

  1. Polymorphisms in isoniazid and prothionamide resistance genes of the Mycobacterium tuberculosis complex.

    PubMed

    Projahn, Michaela; Köser, Claudio U; Homolka, Susanne; Summers, David K; Archer, John A C; Niemann, Stefan

    2011-09-01

    Sequence analyses of 74 strains that encompassed major phylogenetic lineages of the Mycobacterium tuberculosis complex revealed 10 polymorphisms in mshA (Rv0486) and four polymorphisms in inhA (Rv1484) that were not responsible for isoniazid or prothionamide resistance. Instead, some of these mutations were phylogenetically informative. This genetic diversity must be taken into consideration for drug development and for the design of molecular tests for drug resistance. PMID:21709103

  2. Detection of Rifampin Resistance in Mycobacterium tuberculosis by Double Gradient-Denaturing Gradient Gel Electrophoresis

    PubMed Central

    Scarpellini, Paolo; Braglia, Sergio; Carrera, Paola; Cedri, Maura; Cichero, Paola; Colombo, Alessia; Crucianelli, Rosella; Gori, Andrea; Ferrari, Maurizio; Lazzarin, Adriano

    1999-01-01

    We applied double gradient-denaturing gradient gel electrophoresis (DG-DGGE) for the rapid detection of rifampin (RMP) resistance from rpoB PCR products of Mycobacterium tuberculosis isolates and clinical samples. The results of this method were fully concordant with those of DNA sequencing and susceptibility testing analyses. DG-DGGE is a valid alternative to the other methods of detecting mutations for predicting RMP resistance. PMID:10508043

  3. A Mycobacterium tuberculosis IS6110 preferential locus (ipl) for insertion into the genome.

    PubMed Central

    Fang, Z; Forbes, K J

    1997-01-01

    A 267-nucleotide Mycobacterium tuberculosis genomic sequence (ipl, the IS6110 preferential locus) which can harbor the insertion sequence IS6110 at six alternative locations has been identified in some three-quarters of the isolates tested. Only one IS6110 copy was observed at this locus in the ipl::IS6110(+)-containing isolates tested, and all insertions had the same orientation. The implications of this finding for IS6110 fingerprint typing methods is discussed in this work. PMID:9003621

  4. Evaluation of the Epidemiologic Utility of Secondary Typing Methods for Differentiation of Mycobacterium tuberculosis Isolates

    PubMed Central

    Kwara, Awewura; Schiro, Ronald; Cowan, Lauren S.; Hyslop, Newton E.; Wiser, Mark F.; Roahen Harrison, Stephanie; Kissinger, Patricia; Diem, Lois; Crawford, Jack T.

    2003-01-01

    Spoligotyping and mycobacterial interspersed repetitive unit-variable-number tandem repeat analysis (MIRU-VNTR) were evaluated for the ability to differentiate 64 Mycobacterium tuberculosis isolates from 10 IS6110-defined clusters. MIRU-VNTR performed slightly better than spoligotyping in reducing the number of clustered isolates and the sizes of the clusters. All epidemiologically related isolates remained clustered by MIRU-VNTR but not by spoligotyping. PMID:12791904

  5. Molecular Characterization of Isoniazid-Resistant Mycobacterium tuberculosis Clinical Isolates in Lithuania

    PubMed Central

    Bakonyte, Daiva; Baranauskaite, Aurelija; Cicenaite, Jurate; Sosnovskaja, Anaida; Stakenas, Petras

    2003-01-01

    Mutations at codon 315 of the katG gene were detected in 312 of 364 (85.7%) isoniazid-resistant Mycobacterium tuberculosis isolates. Seven of 52 (13.5%) isoniazid-resistant isolates with the wild-type Ser315 codon and 10 of 52 (19.2%) isoniazid-resistant isolates with a mutated katG allele had mutation −15C→T in the promoter of the mabA-inhA operon. PMID:12760887

  6. Polyketides from an Endophytic Aspergillus fumigatus Isolate Inhibit the Growth of Mycobacterium tuberculosis and MRSA.

    PubMed

    Flewelling, Andrew J; Bishop, Amanda I; Johnson, John A; Gray, Christopher A

    2015-10-01

    The crude extract of Aspergillusfumigatus isolate AF3-093A, an endophyte of the brown alga Fucus vesiculosus, showed significant antimicrobial activity in initial bioactivity screens. Bioassay-guided fractionation of the extract led to the isolation of flavipin, chaetoglobosin A and chaetoglobosin B, all of which inhibited the growth of Staphylococcus aureus, methicillin-resistant S. aureus and Mycobacterium tuberculosis H37Ra. The antimycobacterial activity of these compounds has not been previously reported. PMID:26669098

  7. Mycobacterium tuberculosis is the causative agent of tuberculosis in the southern ecological zones of Cameroon, as shown by genetic analysis

    PubMed Central

    2013-01-01

    Background Tuberculosis (TB) is a major cause of mortality and suffering worldwide, with over 95% of TB deaths occurring in low- and middle-income countries. In recent years, molecular typing methods have been widely used in epidemiological studies to aid the control of TB, but this usage has not been the case with many African countries, including Cameroon. The aims of the present investigation were to identify and evaluate the diversity of the Mycobacterium tuberculosis complex (MTBC) isolates circulating in two ecological zones of Cameroon, seven years after the last studies in the West Region, and after the re-organization of the National TB Control Program (NTBCP). These were expected to shed light also on the transmission of TB in the country. The study was conducted from February to July 2009. During this period, 169 patients with symptomatic disease and with sputum cultures that were positive for MTBC were randomly selected for the study from amongst 964 suspected patients in the savannah mosaic zone (West and North West regions) and the tropical rainforest zone (Central region). After culture and diagnosis, DNA was extracted from each of the MTBC isolates and transported to the BecA-ILRI Hub in Nairobi, Kenya for molecular analysis. Methods Genetic characterization was done by mycobacterial interspersed repetitive unit–variable number tandem repeat typing (MIRU-VNTR) and Spoligotyping. Results Molecular analysis showed that all TB cases reported in this study were caused by infections with Mycobacterium tuberculosis (98.8%) and Mycobacterium africanum (M. africanum) (1.2%) respectively. We did not detect any M. bovis. Comparative analyses using spoligotyping revealed that the majority of isolates belong to major clades of M. tuberculosis: Haarlem (7.6%), Latin American-Mediterranean (34.4%) and T clade (26.7%); the remaining isolates (31.3%) where distributed among the minor clades. The predominant group of isolates (34.4%) corresponded to spoligotype 61

  8. Identification of Functional Tat Signal Sequences in Mycobacterium tuberculosis Proteins▿ †

    PubMed Central

    McDonough, Justin A.; McCann, Jessica R.; Tekippe, Erin McElvania; Silverman, Jason S.; Rigel, Nathan W.; Braunstein, Miriam

    2008-01-01

    The twin-arginine translocation (Tat) pathway is a system used by some bacteria to export proteins out from the cytosol to the cell surface or extracellular environment. A functional Tat pathway exists in the important human pathogen Mycobacterium tuberculosis. Identification of the substrates exported by the Tat pathway can help define the role that this pathway plays in the physiology and pathogenesis of M. tuberculosis. Here we used a reporter of Tat export, a truncated β-lactamase, ′BlaC, to experimentally identify M. tuberculosis proteins with functional Tat signal sequences. Of the 13 proteins identified, one lacks the hallmark of a Tat-exported substrate, the twin-arginine dipeptide, and another is not predicted by in silico analysis of the annotated M. tuberculosis genome. Full-length versions of a subset of these proteins were tested to determine if the native proteins are Tat exported. For three proteins, expression in a Δtat mutant of Mycobacterium smegmatis revealed a defect in precursor processing compared to expression in the wild type, indicating Tat export of the full-length proteins. Conversely, two proteins showed no obvious Tat export in M. smegmatis. One of this latter group of proteins was the M. tuberculosis virulence factor phospholipase C (PlcB). Importantly, when tested in M. tuberculosis a different result was obtained and PlcB was exported in a twin-arginine-dependent manner. This suggests the existence of an M. tuberculosis-specific factor(s) for Tat export of a proven virulence protein. It also emphasizes the importance of domains beyond the Tat signal sequence and bacterium-specific factors in determining if a given protein is Tat exported. PMID:18658266

  9. PCR-Based Method To Differentiate the Subspecies of the Mycobacterium tuberculosis Complex on the Basis of Genomic Deletions

    PubMed Central

    Huard, Richard C.; de Oliveira Lazzarini, Luiz Claudio; Butler, W. Ray; van Soolingen, Dick; Ho, John L.

    2003-01-01

    The classical Mycobacterium tuberculosis complex (MtbC) subspecies include Mycobacterium tuberculosis, Mycobacterium africanum (subtypes I and II), Mycobacterium bovis (along with the attenuated M. bovis bacillus Calmette-Guérin [BCG]), and Mycobacterium microti; increasingly recognized MtbC groupings include Mycobacterium bovis subsp. caprae and “Mycobacterium tuberculosis subsp. canettii.” Previous investigations have documented each MtbC subspecies as a source of animal and/or human tuberculosis. However, study of these organisms is hindered by the lack of a single protocol that quickly and easily differentiates all of the MtbC groupings. Towards this end we have developed a rapid, simple, and reliable PCR-based MtbC typing method that makes use of MtbC chromosomal region-of-difference deletion loci. Here, seven primer pairs (which amplify within the loci 16S rRNA, Rv0577, IS1561′, Rv1510, Rv1970, Rv3877/8, and Rv3120) were run in separate but simultaneous reactions. Each primer pair either specifically amplified a DNA fragment of a unique size or failed, depending upon the source mycobacterial DNA. The pattern of amplification products from all of the reactions, visualized by agarose gel electrophoresis, allowed immediate identification either as MtbC composed of M. tuberculosis (or M. africanum subtype II), M. africanum subtype I, M. bovis, M. bovis BCG, M. caprae, M. microti, or “M. canettii” or as a Mycobacterium other than MtbC (MOTT). This MtbC PCR typing panel provides an advanced approach to determine the subspecies of MtbC isolates and to differentiate them from clinically important MOTT species. It has proven beneficial in the management of Mycobacterium collections and may be applied for practical clinical and epidemiological use. PMID:12682155

  10. Molecular basis for the exquisite sensitivity of Mycobacterium tuberculosis to isoniazid.

    PubMed

    Zhang, Y; Dhandayuthapani, S; Deretic, V

    1996-11-12

    The exceptional sensitivity of Mycobacterium tuberculosis to isonicotinic acid hydrazide (INH) lacks satisfactory definition. M. tuberculosis is a natural mutant in oxyR, a central regulator of peroxide stress response. The ahpC gene, which encodes a critical subunit of alkyl hydroperoxide reductase, is one of the targets usually controlled by oxyR in bacteria. Unlike in mycobacterial species less susceptible to INH, the expression of ahpC was below detection limits at the protein level in INH-sensitive M. tuberculosis and Mycobacterium bovis strains. In contrast, AhpC was detected in several series of isogenic INH-resistant (INHr) derivatives. In a demonstration of the critical role of ahpC in sensitivity to INH, insertional inactivation of ahpC on the chromosome of Mycobacterium smegmatis, a species naturally insensitive to INH, dramatically increased its susceptibility to this compound. These findings suggest that AhpC counteracts the action of INH and that the levels of its expression may govern the intrinsic susceptibility of mycobacteria to this front-line antituberculosis drug. PMID:8917570

  11. Dehalogenation of haloalkanes by Mycobacterium tuberculosis H37Rv and other mycobacteria

    SciTech Connect

    Jesenska, A.; Sedlacek, I.; Damborsky, J.

    2000-01-01

    Haloalkane dehalogenases convert haloalkanes to their corresponding alcohols by a hydrolytic mechanism. To date, various haloalkane dehalogenases have been isolated from bacteria colonizing environments that are contaminated with halogenated compounds. A search of current databases with the sequences of these known haloalkane dehalogenases revealed the presence of three different genes encoding putative haloalkane dehalogenases in the genome of the human parasite Mycobacterium tuberculosis H37Rv. The ability of M. tuberculosis and several other mycobacterial strains to dehalogenate haloaliphatic compounds was therefore studied. Intact cells of M. tuberculosis H37Rv were found to dehalogenate 1-chlorobutane, 1-chlorodecane, 1-bromobutane, and 1,2-dibromoethane. Nine isolates of mycobacteria from clinical material and four strains from a collection of microorganisms were found to be capable of dehalogenating 1,2-dibromoethane. Crude extracts prepared from two of these strains, Mycobacterium avium MU1 and Mycobacterium smegmatis CCM 4622, showed broad substrate specificity toward a number of halogenated substrates. Dehalogenase activity in the absence of oxygen and the identification of primary alcohols as the products of the reaction suggest a hydrolytic dehalogenation mechanism. The presence of dehalogenases in bacterial isolates from clinical material, including the species colonizing both animal tissues and free environment, indicates a possible role of parasitic microorganisms in the distribution of degradation genes in the environment.

  12. An Acyl-CoA Synthetase in Mycobacterium tuberculosis Involved in Triacylglycerol Accumulation during Dormancy

    PubMed Central

    Daniel, Jaiyanth; Sirakova, Tatiana; Kolattukudy, Pappachan

    2014-01-01

    Latent infection with dormant Mycobacterium tuberculosis is one of the major reasons behind the emergence of drug-resistant strains of the pathogen worldwide. In its dormant state, the pathogen accumulates lipid droplets containing triacylglycerol synthesized from fatty acids derived from host lipids. In this study, we show that Rv1206 (FACL6), which is annotated as an acyl-CoA synthetase and resembles eukaryotic fatty acid transport proteins, is able to stimulate fatty acid uptake in E. coli cells. We show that purified FACL6 displays acyl-coenzyme A synthetase activity with a preference towards oleic acid, which is one of the predominant fatty acids in host lipids. Our results indicate that the expression of FACL6 protein in Mycobacterium tuberculosis is significantly increased during in vitro dormancy. The facl6-deficient Mycobacterium tuberculosis mutant displayed a diminished ability to synthesize acyl-coenzyme A in cell-free extracts. Furthermore, during in vitro dormancy, the mutant synthesized lower levels of intracellular triacylglycerol from exogenous fatty acids. Complementation partially restored the lost function. Our results suggest that FACL6 modulates triacylglycerol accumulation as the pathogen enters dormancy by activating fatty acids. PMID:25490545

  13. Novel genetic polymorphisms that further delineate the phylogeny of the Mycobacterium tuberculosis complex.

    PubMed

    Huard, Richard C; Fabre, Michel; de Haas, Petra; Lazzarini, Luiz Claudio Oliveira; van Soolingen, Dick; Cousins, Debby; Ho, John L

    2006-06-01

    In a previous report, we described a PCR protocol for the differentiation of the various species of the Mycobacterium tuberculosis complex (MTC) on the basis of genomic deletions (R. C. Huard, L. C. de Oliveira Lazzarini, W. R. Butler, D. van Soolingen, and J. L. Ho, J. Clin. Microbiol. 41:1637-1650, 2003). That report also provided a broad cross-comparison of several previously identified, phylogenetically relevant, long-sequence and single-nucleotide polymorphisms (LSPs and SNPs, respectively). In the present companion report, we expand upon the previous work (i) by continuing the evaluation of known MTC phylogenetic markers in a larger collection of tubercle bacilli (n = 125), (ii) by evaluating additional recently reported MTC species-specific and interspecific polymorphisms, and (iii) by describing the identification and distribution of a number of novel LSPs and SNPs. Notably, new genomic deletions were found in various Mycobacterium tuberculosis strains, new species-specific SNPs were identified for "Mycobacterium canettii," Mycobacterium microti, and Mycobacterium pinnipedii, and, for the first time, intraspecific single-nucleotide DNA differences were discovered for the dassie bacillus, the oryx bacillus, and the two Mycobacterium africanum subtype I variants. Surprisingly, coincident polymorphisms linked one M. africanum subtype I genotype with the dassie bacillus and M. microti with M. pinnipedii, thereby suggesting closer evolutionary ties within each pair of species than had been previously thought. Overall, the presented data add to the genetic definitions of several MTC organisms as well as fine-tune current models for the evolutionary history of the MTC. PMID:16740934

  14. Co-infection of Mycobacterium tuberculosis and Mycobacterium leprae in human archaeological samples: a possible explanation for the historical decline of leprosy

    PubMed Central

    Donoghue, Helen D.; Marcsik, Antónia; Matheson, Carney; Vernon, Kim; Nuorala, Emilia; Molto, Joseph E.; Greenblatt, Charles L.; Spigelman, Mark

    2005-01-01

    Both leprosy and tuberculosis were prevalent in Europe during the first millennium but thereafter leprosy declined. It is not known why this occurred, but one suggestion is that cross-immunity protected tuberculosis patients from leprosy. To investigate any relationship between the two diseases, selected archaeological samples, dating from the Roman period to the thirteenth century, were examined for both Mycobacterium leprae and Mycobacterium tuberculosis DNA, using PCR. The work was carried out and verified in geographically separate and independent laboratories. Several specimens with palaeopathological signs of leprosy were found to contain DNA from both pathogens, indicating that these diseases coexisted in the past. We suggest that the immunological changes found in multi-bacillary leprosy, in association with the socio-economic impact on those suffering from the disease, led to increased mortality from tuberculosis and therefore to the historical decline in leprosy. PMID:15734693

  15. The Polyketide Pks1 Contributes to Biofilm Formation in Mycobacterium tuberculosis

    PubMed Central

    Pang, Jennifer M.; Layre, Emilie; Sweet, Lindsay; Sherrid, Ashley; Moody, D. Branch; Ojha, Anil

    2012-01-01

    Infections caused by biofilms are abundant and highly persistent, displaying phenotypic resistance to high concentrations of antimicrobials and modulating host immune systems. Tuberculosis (TB), caused by Mycobacterium tuberculosis, shares these qualities with biofilm infections. To identify genetic determinants of biofilm formation in M. tuberculosis, we performed a small-scale transposon screen using an in vitro pellicle biofilm assay. We identified five M. tuberculosis mutants that were reproducibly attenuated for biofilm production relative to that of the parent strain H37Rv. One of the most attenuated mutants is interrupted in pks1, a polyketide synthase gene. When fused with pks15, as in some M. tuberculosis isolates, pks1 contributes to synthesis of the immunomodulatory phenolic glycolipids (PGLs). However, in strains such as H37Rv with split pks15 and pks1 loci, PGL is not produced and pks1 has no previously defined role. We showed that pks1 complementation restores biofilm production independently of the known role of pks1 in PGL synthesis. We also assessed the relationship among biofilm formation, the pks15/1 genotype, and M. tuberculosis phylogeography. A global survey of M. tuberculosis clinical isolates revealed surprising sequence variability in the pks15/1 locus and substantial variation in biofilm phenotypes. Our studies identify novel M. tuberculosis genes that contribute to biofilm production, including pks1. In addition, we find that the ability to make pellicle biofilms is common among M. tuberculosis isolates from throughout the world, suggesting that this trait is relevant to TB propagation or persistence. PMID:22123254

  16. Human humoral responses to antigens of Mycobacterium tuberculosis: immunodominance of high-molecular-mass antigens.

    PubMed Central

    Laal, S; Samanich, K M; Sonnenberg, M G; Zolla-Pazner, S; Phadtare, J M; Belisle, J T

    1997-01-01

    The selection of antigens of Mycobacterium tuberculosis for most studies of humoral responses in tuberculosis patients has been restricted to molecules that were either immunodominant in immunized animals or amenable to biochemical purification rather than those that were reactive with the human immune system. Delineation of antigens that elicit humoral responses during the natural course of disease progression in humans has been hindered by the presence of cross-reactive antibodies to conserved regions on ubiquitous prokaryotic antigens in sera from healthy individuals and tuberculosis patients. The levels of cross-reactive antibodies in the sera were reduced by preadsorption with Escherichia coli lysates, prior to studying their reactivity against a large panel of M. tuberculosis antigens to which the human immune system may be exposed during natural infection and disease. Thus, reactivity against pools of secreted, cellular, and cell wall-associated antigens of M. tuberculosis was assessed by an enzyme-linked immunosorbent assay (ELISA). Initial results suggested that the secreted protein preparation contained antigens most frequently recognized by the humoral responses of pulmonary tuberculosis patients. The culture filtrate proteins were subsequently size fractionated by preparative polyacrylamide gel electrophoresis, characterized by reaction with murine monoclonal antibodies to known antigens of M. tuberculosis by an ELISA, and assessed for reactivity with tuberculous and nontuberculous sera. Results show that a secreted antigen of 88 kDa elicits a strong antibody response in a high percentage of patients with pulmonary tuberculosis. This and other antigens identified on the basis of their reactivity with patient sera may prove useful for developing serodiagnosis for tuberculosis. PMID:9008280

  17. MntR(Rv2788): a transcriptional regulator that controls manganese homeostasis in Mycobacterium tuberculosis.

    PubMed

    Pandey, Ruchi; Russo, Riccardo; Ghanny, Saleena; Huang, Xiaojuan; Helmann, John; Rodriguez, G Marcela

    2015-12-01

    The pathogenic mycobacterium Mycobacterium tuberculosis encodes two members of the DtxR/MntR family of metalloregulators, IdeR and SirR. IdeR represses gene expression in response to ferrous iron, and we here demonstrate that SirR (Rv2788), although also annotated as an iron-dependent repressor, functions instead as a manganese-dependent transcriptional repressor and is therefore renamed MntR. MntR regulates transporters that promote manganese import and genes that respond to metal ion deficiency such as the esx3 system. Repression of manganese import by MntR is essential for survival of M. tuberculosis under conditions of high manganese availability, but mntR is dispensable during infection. In contrast, manganese import by MntH and MntABCD was found to be indispensable for replication of M. tuberculosis in macrophages. These results suggest that manganese is limiting in the host and that interfering with import of this essential metal may be an effective strategy to attenuate M. tuberculosis. PMID:26337157

  18. Characterization of Mycobacterium tuberculosis complex direct repeat sequence for use in cycling probe reaction.

    PubMed Central

    Beggs, M L; Cave, M D; Marlowe, C; Cloney, L; Duck, P; Eisenach, K D

    1996-01-01

    Cycling probe technology (CPT) is a unique and simple method for the detection of specific target sequences. CPT utilizes a chimeric DNA-RNA-DNA probe providing an RNase H-sensitive scissile linkage when bound to a complementary target sequence. For this study a diagnostic assay based on CPT was developed for the detection of the 36-bp direct repeat (DR) region in Mycobacterium tuberculosis. To determine the feasibility of using the DR for detecting M. tuberculosis by CPT, a wide variety of mycobacteria were tested by Southern blot hybridization with three DR probes to verify their specificity. The entire DR region of Mycobacterium bovis 401 was sequenced, and the data were used to design a PCR assay that would allow us to estimate the number of DRs present in a variety of strains. A CPT assay which uses a probe complementary to the DR region was developed and evaluated with synthetic targets and genomic DNA from mycobacteria. In summary, the 36-bp DR provides an attractive target for detecting M. tuberculosis because the sequence is present in high copy numbers in the genome, is specific for the M. tuberculosis complex, and is found in strains that lack IS6110. PMID:8940435

  19. Dynamics of Mycobacterium and bovine tuberculosis in a Human-Buffalo Population

    PubMed Central

    Hassan, A. S.; Garba, S. M.; Gumel, A. B.; Lubuma, J. M.-S.

    2014-01-01

    A new model for the transmission dynamics of Mycobacterium tuberculosis and bovine tuberculosis in a community, consisting of humans and African buffalos, is presented. The buffalo-only component of the model exhibits the phenomenon of backward bifurcation, which arises due to the reinfection of exposed and recovered buffalos, when the associated reproduction number is less than unity. This model has a unique endemic equilibrium, which is globally asymptotically stable for a special case, when the reproduction number exceeds unity. Uncertainty and sensitivity analyses, using data relevant to the dynamics of the two diseases in the Kruger National Park, show that the distribution of the associated reproduction number is less than unity (hence, the diseases would not persist in the community). Crucial parameters that influence the dynamics of the two diseases are also identified. Both the buffalo-only and the buffalo-human model exhibit the same qualitative dynamics with respect to the local and global asymptotic stability of their respective disease-free equilibrium, as well as with respect to the backward bifurcation phenomenon. Numerical simulations of the buffalo-human model show that the cumulative number of Mycobacterium tuberculosis cases in humans (buffalos) decreases with increasing number of bovine tuberculosis infections in humans (buffalo). PMID:25254065

  20. Evaluation of spoligotyping in a study of the transmission of Mycobacterium tuberculosis.

    PubMed Central

    Goguet de la Salmonière, Y O; Li, H M; Torrea, G; Bunschoten, A; van Embden, J; Gicquel, B

    1997-01-01

    Spoligotyping (for spacer oligotyping) is an easy, economical, and rapid way of typing Mycobacterium tuberculosis complex strains with the DR spacer markers (J. Kamerbeek et al., J. Clin. Microbiol. 35:907-914, 1997; D. van Soolingen et al., 33:3234-3248, 1995). The stability of the markers was demonstrated by showing that all the Mycobacterium bovis BCG strains tested gave the same spoligotyping pattern. None of the 42 atypical mycobacterial strains tested gave a spoligotyping signal, indicating the specificity of the technique for M. tuberculosis complex. The utility of the spoligotyping method was demonstrated by analyzing 106 isolates of M. tuberculosis obtained over 1 year in three Paris hospitals. The results obtained by this technique were compared to those obtained by Torrea et al. (G. Torrea et al., J. Clin. Microbiol. 34:1043-1049, 1996) by IS6110-based restriction fragment length polymorphism (RFLP) analysis. Strains from patients with epidemiological relationships that were in the same IS6110-RFLP cluster were also in the same spoligotyping group. Spoligotyping was more discriminative than RFLP analysis for strains with one or two copies of IS6110. RFLP analysis did not discriminate between the nine strains with one or two IS6110 bands with no known epidemiological relation, whereas spoligotyping distinguished between eight different types. IS6I10-RFLP analysis split some of the spoligotyping clusters, particularly when the IS6110 copy number was high. Therefore, we propose a strategy for typing M. tuberculosis strains in which both markers are used. PMID:9276389

  1. Development of a murine mycobacterial growth inhibition assay for evaluating vaccines against Mycobacterium tuberculosis.

    PubMed

    Parra, Marcela; Yang, Amy L; Lim, JaeHyun; Kolibab, Kristopher; Derrick, Steven; Cadieux, Nathalie; Perera, Liyanage P; Jacobs, William R; Brennan, Michael; Morris, Sheldon L

    2009-07-01

    The development and characterization of new tuberculosis (TB) vaccines has been impeded by the lack of reproducible and reliable in vitro assays for measuring vaccine activity. In this study, we developed a murine in vitro mycobacterial growth inhibition assay for evaluating TB vaccines that directly assesses the capacity of immune splenocytes to control the growth of Mycobacterium tuberculosis within infected macrophages. Using this in vitro assay, protective immune responses induced by immunization with five different types of TB vaccine preparations (Mycobacterium bovis BCG, an attenuated M. tuberculosis mutant strain, a DNA vaccine, a modified vaccinia virus strain Ankara [MVA] construct expressing four TB antigens, and a TB fusion protein formulated in adjuvant) can be detected. Importantly, the levels of vaccine-induced mycobacterial growth-inhibitory responses seen in vitro after 1 week of coculture correlated with the protective immune responses detected in vivo at 28 days postchallenge in a mouse model of pulmonary tuberculosis. In addition, similar patterns of cytokine expression were evoked at day 7 of the in vitro culture by immune splenocytes taken from animals immunized with the different TB vaccines. Among the consistently upregulated cytokines detected in the immune cocultures are gamma interferon, growth differentiation factor 15, interleukin-21 (IL-21), IL-27, and tumor necrosis factor alpha. Overall, we have developed an in vitro functional assay that may be useful for screening and comparing new TB vaccine preparations, investigating vaccine-induced protective mechanisms, and assessing manufacturing issues, including product potency and stability. PMID:19458207

  2. The Association between Mycobacterium Tuberculosis Genotype and Drug Resistance in Peru

    PubMed Central

    Grandjean, Louis; Iwamoto, Tomotada; Lithgow, Anna; Gilman, Robert H; Arikawa, Kentaro; Nakanishi, Noriko; Martin, Laura; Castillo, Edith; Alarcon, Valentina; Coronel, Jorge; Solano, Walter; Aminian, Minoo; Guezala, Claudia; Rastogi, Nalin; Couvin, David; Sheen, Patricia; Zimic, Mirko; Moore, David AJ

    2015-01-01

    Background The comparison of Mycobacterium tuberculosis bacterial genotypes with phenotypic, demographic, geospatial and clinical data improves our understanding of how strain lineage influences the development of drug-resistance and the spread of tuberculosis. Methods To investigate the association of Mycobacterium tuberculosis bacterial genotype with drug-resistance. Drug susceptibility testing together with genotyping using both 15-loci MIRU-typing and spoligotyping, was performed on 2,139 culture positive isolates, each from a different patient in Lima, Peru. Demographic, geospatial and socio-economic data were collected using questionnaires, global positioning equipment and the latest national census. Results The Latin American Mediterranean (LAM) clade (OR 2.4, p<0.001) was significantly associated with drug-resistance and alone accounted for more than half of all drug resistance in the region. Previously treated patients, prisoners and genetically clustered cases were also significantly associated with drug-resistance (OR's 2.5, 2.4 and 1.8, p<0.001, p<0.05, p<0.001 respectively). Conclusions Tuberculosis disease caused by the LAM clade was more likely to be drug resistant independent of important clinical, genetic and socio-economic confounding factors. Explanations for this include; the preferential co-evolution of LAM strains in a Latin American population, a LAM strain bacterial genetic background that favors drug-resistance or the "founder effect" from pre-existing LAM strains disproportionately exposed to drugs. PMID:25984723

  3. Use of Roche AMPLICOR Mycobacterium tuberculosis PCR in Early Diagnosis of Tuberculous Meningitis

    PubMed Central

    Bonington, Alec; Strang, J. I. George; Klapper, Paul E.; Hood, Steven V.; Rubombora, William; Penny, Miranda; Willers, Rose; Wilkins, Edmund G. L.

    1998-01-01

    Several nucleic acid-based amplification tests are available for the detection of Mycobacterium tuberculosis, but few data are available on their use in the diagnosis of tuberculous meningitis (TBM). We performed a prospective study to assess the Roche AMPLICOR Mycobacterium tuberculosis PCR test (TB AMPLICOR) for use in the diagnosis of TBM and compared it with direct Ziehl-Neelsen staining of smears, radiometric culture for M. tuberculosis, and clinical and cerebrospinal fluid (CSF) findings. Eighty-three CSF specimens collected from 69 patients with suspected meningitis in South Africa were tested by TB AMPLICOR. On the basis of clinical and laboratory findings, 40 of these patients were treated for TBM and 29 patients were not treated for TBM. Ten CSF samples from 10 patients were positive by TB AMPLICOR. Seven of these 10 patients were classified as having definite TBM, 2 were classified as having probable TBM, and 1 was classified as having possible TBM. The sensitivity of TB AMPLICOR for detecting cases of definite and probable TBM in patients from whom CSF specimens had been collected less than 10 days into antituberculosis treatment was 60.0%. Specimens from all 29 patients not treated for TBM were negative by the TB AMPLICOR, giving a 100% specificity. TB AMPLICOR is therefore more sensitive than the combination of Ziehl-Neelsen staining of smears and radiometric culture for M. tuberculosis and is a rapid and highly specific diagnostic test for TBM. PMID:9574686

  4. Mycobacterium sherrisii Lung Infection in a Brazilian Patient with Silicosis and a History of Pulmonary Tuberculosis

    PubMed Central

    de Oliveira Abrão, Carolina; de Araújo Filho, João Alves

    2015-01-01

    Nontuberculous mycobacteria (NTM) diseases became relevant with the emergence and spread of HIV and are also related to lung infection in non-HIV individuals with structural lung diseases. Mycobacterium sherrisii is a NTM first characterized in 2004. Only a few cases have been reported. The aim of this case report is to describe the first detailed case of infection with M. sherrisii in a patient with silicosis and history of pulmonary tuberculosis. A 50-year-old HIV-negative white male, previous smoker, with silicosis and a history of treated pulmonary tuberculosis developed a worsening of cough and expectoration pattern, and two sputum samples were positive for acid-fast bacilli. Presumptive treatment for pulmonary tuberculosis was initiated with rifampin, isoniazid, pyrazinamide, and ethambutol, but, at month 5 of treatment, despite correct medication intake and slight improvement of symptoms, sputum bacilloscopy remained positive. Sputum cultures were positive Mycobacterium sherrisii. Treatment regimen was altered to streptomycin (for 2 months), ethambutol, clarithromycin, rifabutin, and trimethoprim-sulfamethoxazole. M. sherrisii should be considered a possible etiological agent of lung infections in patients with pneumoconiosis and history of tuberculosis. PMID:26557395

  5. EBP50 induces apoptosis in macrophages by upregulating nitric oxide production to eliminate intracellular Mycobacterium tuberculosis

    PubMed Central

    Guo, Yang; Deng, Yating; Huang, Zikun; Luo, Qing; Peng, Yiping; Chen, Jie; Jiang, Hong; Ye, Jianqing; Li, Junming

    2016-01-01

    Mycobacterium bovis BCG is known to have the capacity to inhibit the positioning of iNOS on BCG-containing phagosomes by interfering with EBP50, a scaffolding protein that controls the recruitment of inducible nitric oxide synthase (iNOS) at the vicinity of phagosomes in macrophages. However, knockdown of the expression of EBP50 still facilitates the intracellular survival of BCG, which suggested that EBP50 may have some other unknown antimycobacterial properties. In this study we show that overexpression of EBP50 by a recombinant lentivirus had no effect on the iNOS recruitment to M.tuberculosis-containing phagosomes, but significantly promoted the elimination of intracellular M.tuberculosis. We revealed in the present study that the enhancement of intracellular killing to M. tuberculosis upon EBP50 overexpression was due to the increased level of apoptosis in macrophages. We showed that EBP50 overexpression significantly increased the expression of iNOS and generation of nitric oxide (NO), and EBP50-induced apoptosis was NO-dependent and mediated by Bax and caspase-3. We found that M. tuberculosis decreases while Mycobacterium smegmatis increases the expression of EBP50 in RAW264.7 cells, which suggested that virulent mycobacteria are capable of modulating the antimycobacterial properties of macrophages by inhibiting the expression and interfering with the function of EBP50. PMID:26729618

  6. Evidence for the presence of a phosphatidylinositol anchor on the lipoarabinomannan and lipomannan of Mycobacterium tuberculosis.

    PubMed

    Hunter, S W; Brennan, P J

    1990-06-01

    The recent availability (Hunter, S.W., Gaylord, H., and Brennan, P.J. (1986) J. Biol. Chem. 261, 12345-12351) of the well known arabinomannan of Mycobacterium leprae and Mycobacterium tuberculosis as the pure native lipoarabinomannan has resulted in its implication in key aspects of the immunopathogenesis of leprosy and tuberculosis. We had indicated that the lipid moiety of lipoarabinomannan is probably based on a diacylglycerol unit in that glycerol and the two fatty acids, hexadecanoate and 10-methyloctadecanoate, were identified. In addition, lipoarabinomannan was also shown to contain myo-inositol 1-phosphate. Evidence is now presented, based on selective radiolabeling and analysis of various cleavage fragments, that the inositol phosphate exists as both an alkalilable phosphodiester and as part of a phosphatidylinositol "membrane anchor." The mannan of M. tuberculosis was also isolated as the native lipomannan. It also apparently contains a phosphatidylinositol unit but is devoid of the alkali-labile inositol phosphate residues. These lipopolysaccharides are apparently multiglycosylated versions of the well known myocobacterial mannosyl phosphatidylinositols and are prokaryotic versions of the growing list of phosphatidylinositol-anchored macromolecules. Immunogold labeling demonstrates that lipoarabinomannan is a true antigenic capsular or extracellular product of M. tuberculosis. The presence of a phosphatidylinositol residue on lipoarabinomannan may explain its interaction with macrophage membranes and role in mycobacterial pathogenesis. PMID:2111816

  7. Widespread Environmental Contamination with Mycobacterium tuberculosis Complex Revealed by a Molecular Detection Protocol.

    PubMed

    Santos, Nuno; Santos, Catarina; Valente, Teresa; Gortázar, Christian; Almeida, Virgílio; Correia-Neves, Margarida

    2015-01-01

    Environmental contamination with Mycobacterium tuberculosis complex (MTC) has been considered crucial for bovine tuberculosis persistence in multi-host-pathogen systems. However, MTC contamination has been difficult to detect due to methodological issues. In an attempt to overcome this limitation we developed an improved protocol for the detection of MTC DNA. MTC DNA concentration was estimated by the Most Probable Number (MPN) method. Making use of this protocol we showed that MTC contamination is widespread in different types of environmental samples from the Iberian Peninsula, which supports indirect transmission as a contributing mechanism for the maintenance of bovine tuberculosis in this multi-host-pathogen system. The proportion of MTC DNA positive samples was higher in the bovine tuberculosis-infected than in presumed negative area (0.32 and 0.18, respectively). Detection varied with the type of environmental sample and was more frequent in sediment from dams and less frequent in water also from dams (0.22 and 0.05, respectively). The proportion of MTC-positive samples was significantly higher in spring (p<0.001), but MTC DNA concentration per sample was higher in autumn and lower in summer. The average MTC DNA concentration in positive samples was 0.82 MPN/g (CI95 0.70-0.98 MPN/g). We were further able to amplify a DNA sequence specific of Mycobacterium bovis/caprae in 4 environmental samples from the bTB-infected area. PMID:26561038

  8. Widespread Environmental Contamination with Mycobacterium tuberculosis Complex Revealed by a Molecular Detection Protocol

    PubMed Central

    Santos, Nuno; Santos, Catarina; Valente, Teresa; Gortázar, Christian; Almeida, Virgílio; Correia-Neves, Margarida

    2015-01-01

    Environmental contamination with Mycobacterium tuberculosis complex (MTC) has been considered crucial for bovine tuberculosis persistence in multi-host-pathogen systems. However, MTC contamination has been difficult to detect due to methodological issues. In an attempt to overcome this limitation we developed an improved protocol for the detection of MTC DNA. MTC DNA concentration was estimated by the Most Probable Number (MPN) method. Making use of this protocol we showed that MTC contamination is widespread in different types of environmental samples from the Iberian Peninsula, which supports indirect transmission as a contributing mechanism for the maintenance of bovine tuberculosis in this multi-host-pathogen system. The proportion of MTC DNA positive samples was higher in the bovine tuberculosis-infected than in presumed negative area (0.32 and 0.18, respectively). Detection varied with the type of environmental sample and was more frequent in sediment from dams and less frequent in water also from dams (0.22 and 0.05, respectively). The proportion of MTC-positive samples was significantly higher in spring (p<0.001), but MTC DNA concentration per sample was higher in autumn and lower in summer. The average MTC DNA concentration in positive samples was 0.82 MPN/g (CI95 0.70–0.98 MPN/g). We were further able to amplify a DNA sequence specific of Mycobacterium bovis/caprae in 4 environmental samples from the bTB-infected area. PMID:26561038

  9. Molecular modeling of Mycobacterium tuberculosis dUTpase: docking and catalytic mechanism studies.

    PubMed

    Ramalho, Teodorico C; Caetano, Melissa S; Josa, Daniela; Luz, Gustavo P; Freitas, Elisangela A; da Cunha, Elaine F F

    2011-06-01

    Mycobacterium tuberculosis is a leading cause of infectious disease in the world today. This outlook is aggravated by a growing number of M. tuberculosis infections in individuals who are immunocompromised as a result of HIV infections. Thus, new and more potent anti-TB agents are necessary. Therefore, dUTpase was selected as a target enzyme to combat M. tuberculosis. In this work, molecular modeling methods involving docking and QM/MM calculations were carried out to investigate the binding orientation and predict binding affinities of some potential dUTpase inhibitors. Our results suggest that the best potential inhibitor investigated, among the compounds studied in this work, is the compound dUPNPP. Regarding the reaction mechanism, we concluded that the decisive stage for the reaction is the stage 1. Furthermore, it was also observed that the compounds with a -1 electrostatic charge presented lower activation energy in relation to the compounds with a -2 charge. PMID:21469751

  10. Encapsulation of moxifloxacin within poly(butyl cyanoacrylate) nanoparticles enhances efficacy against intracellular Mycobacterium tuberculosis.

    PubMed

    Kisich, K O; Gelperina, S; Higgins, M P; Wilson, S; Shipulo, E; Oganesyan, E; Heifets, L

    2007-12-10

    Macrophages in the lungs are the most important cell type supporting replication of Mycobacterium tuberculosis in humans. The objective of this study was to investigate whether the effect of moxifloxacin against M. tuberculosis residing in macrophages could be improved by encapsulation of the drug in the biodegradable nanoparticles, which are known to accumulate in macrophages upon intravenous administration. To accomplish this, moxifloxacin was encapsulated in poly(butyl cyanoacrylate) (PBCA) nanoparticles. Encapsulated moxifloxacin accumulated in macrophages approximately three-fold times more efficiently than the free drug, and was detected in the cells for at least six times longer than free moxifloxacin at the same extracellular concentration. Inhibition of intracellular M. tuberculosis growth with encapsulated moxifloxacin was achieved at the concentration of 0.1microg/ml, whereas the same effect with free MX required a concentration of 1microg/ml. Nanoparticles observed within the macrophage cytoplasm were distributed throughout the cytoplasm, sometimes in the vicinity of intracellular bacteria. PMID:17624699

  11. Heminested inverse PCR for IS6110 fingerprinting of Mycobacterium tuberculosis strains.

    PubMed Central

    Patel, S; Wall, S; Saunders, N A

    1996-01-01

    A heminested inverse PCR (HIP) for the amplification of sequences flanking the Mycobacterium tuberculosis insertion sequence IS6110 has been developed. The method depends upon primers that anneal to IS6110 at sites between its 5' end and the closest BsrFI site. The accuracy of HIP was demonstrated by the amplification of sequences within plasmid constructs carrying one or two copies of the insertion sequence IS986 in different orientations. The identities of the amplicons produced from strains carrying a single copy of IS6110 were verified by nucleotide sequencing. Analyses of 204 M. tuberculosis strains including those involved in outbreaks showed that IS6110 HIP is highly discriminatory and reproducible. HIP fingerprinting of these 204 strains generated 136 distinct types, and its discriminatory power was equivalent to that of standard restriction fragment length polymorphism analysis. The method is therefore of value for the rapid fingerprinting of M. tuberculosis strains for epidemiological purposes. PMID:8784570

  12. Cloning and characterization of the aroA gene from Mycobacterium tuberculosis.

    PubMed Central

    Garbe, T; Jones, C; Charles, I; Dougan, G; Young, D

    1990-01-01

    The aroA gene from Mycobacterium tuberculosis has been cloned by complementation of an aroA mutant of Escherichia coli after lysogenization with a recombinant DNA library in the lambda gt11 vector. Detailed characterization of the M. tuberculosis aroA gene by nucleotide sequencing and by immunochemical analysis of the expressed product indicates that it encodes a 5-enolpyruvylshikimate-3-phosphate synthase that is structurally related to analogous enzymes from other bacterial, fungal, and plant sources. The potential use of the cloned gene in construction of genetically defined mutant strains of M. tuberculosis by gene replacement is proposed as a novel approach to the rational attenuation of mycobacterial pathogens and the possible development of new antimycobacterial vaccines. Images PMID:2123856

  13. Current prospects of synthetic curcumin analogs and chalcone derivatives against mycobacterium tuberculosis.

    PubMed

    Bukhari, Syed Nasir Abbas; Franzblau, Scott G; Jantan, Ibrahim; Jasamai, Malina

    2013-11-01

    Tuberculosis, caused by Mycobacterium tuberculosis, is amongst the foremost infectious diseases. Treatment of tuberculosis is a complex process due to various factors including a patient's inability to persevere with a combined treatment regimen, the difficulty in eradicating the infection in immune-suppressed patients, and multidrug resistance (MDR). Extensive research circumscribing molecules to counteract this disease has led to the identification of many inhibitory small molecules. Among these are chalcone derivatives along with curcumin analogs. In this review article, we summarize the reported literature regarding anti tubercular activity of chalcone derivatives and synthetic curcumin analogs. Our goal is to provide an analysis of research to date in order to facilitate the synthesis of superior antitubercular chalcone derivatives and curcumin analogs. PMID:23305394

  14. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence.

    PubMed

    Cole, S T; Brosch, R; Parkhill, J; Garnier, T; Churcher, C; Harris, D; Gordon, S V; Eiglmeier, K; Gas, S; Barry, C E; Tekaia, F; Badcock, K; Basham, D; Brown, D; Chillingworth, T; Connor, R; Davies, R; Devlin, K; Feltwell, T; Gentles, S; Hamlin, N; Holroyd, S; Hornsby, T; Jagels, K; Krogh, A; McLean, J; Moule, S; Murphy, L; Oliver, K; Osborne, J; Quail, M A; Rajandream, M A; Rogers, J; Rutter, S; Seeger, K; Skelton, J; Squares, R; Squares, S; Sulston, J E; Taylor, K; Whitehead, S; Barrell, B G

    1998-06-11

    Countless millions of people have died from tuberculosis, a chronic infectious disease caused by the tubercle bacillus. The complete genome sequence of the best-characterized strain of Mycobacterium tuberculosis, H37Rv, has been determined and analysed in order to improve our understanding of the biology of this slow-growing pathogen and to help the conception of new prophylactic and therapeutic interventions. The genome comprises 4,411,529 base pairs, contains around 4,000 genes, and has a very high guanine + cytosine content that is reflected in the biased amino-acid content of the proteins. M. tuberculosis differs radically from other bacteria in that a very large portion of its coding capacity is devoted to the production of enzymes involved in lipogenesis and lipolysis, and to two new families of glycine-rich proteins with a repetitive structure that may represent a source of antigenic variation. PMID:9634230

  15. Reconstruction and topological characterization of the sigma factor regulatory network of Mycobacterium tuberculosis.

    PubMed

    Chauhan, Rinki; Ravi, Janani; Datta, Pratik; Chen, Tianlong; Schnappinger, Dirk; Bassler, Kevin E; Balázsi, Gábor; Gennaro, Maria Laura

    2016-01-01

    Accessory sigma factors, which reprogram RNA polymerase to transcribe specific gene sets, activate bacterial adaptive responses to noxious environments. Here we reconstruct the complete sigma factor regulatory network of the human pathogen Mycobacterium tuberculosis by an integrated approach. The approach combines identification of direct regulatory interactions between M. tuberculosis sigma factors in an E. coli model system, validation of selected links in M. tuberculosis, and extensive literature review. The resulting network comprises 41 direct interactions among all 13 sigma factors. Analysis of network topology reveals (i) a three-tiered hierarchy initiating at master regulators, (ii) high connectivity and (iii) distinct communities containing multiple sigma factors. These topological features are likely associated with multi-layer signal processing and specialized stress responses involving multiple sigma factors. Moreover, the identification of overrepresented network motifs, such as autoregulation and coregulation of sigma and anti-sigma factor pairs, provides structural information that is relevant for studies of network dynamics. PMID:27029515

  16. Rv1894c Is a Novel Hypoxia-Induced Nitronate Monooxygenase Required for Mycobacterium tuberculosis Virulence

    PubMed Central

    Klinkenberg, Lee G.; Karakousis, Petros C.

    2013-01-01

    Tuberculosis is difficult to cure, requiring a minimum of 6 months of treatment with multiple antibiotics. Small numbers of organisms are able to tolerate the antibiotics and persist in the lungs of infected humans, but they still require some metabolic activity to survive. We studied the role of the hypoxia-induced Rv1894c gene in Mycobacterium tuberculosis virulence in guinea pigs, which develop hypoxic, necrotic granulomas histologically resembling those in humans and found this gene to be necessary for full bacillary growth and survival. We characterized the function of the encoded enzyme as a nitronate monooxygenase, which is needed to prevent the buildup of toxic products during hypoxic metabolism and is negatively regulated by the transcriptional repressor KstR. Future studies will focus on developing small-molecule inhibitors that target Rv1894c and its homologs, with the goal of killing persistent bacteria, thereby shortening the time needed to treat tuberculosis. PMID:23408846

  17. Reconstruction and topological characterization of the sigma factor regulatory network of Mycobacterium tuberculosis

    PubMed Central

    Chauhan, Rinki; Ravi, Janani; Datta, Pratik; Chen, Tianlong; Schnappinger, Dirk; Bassler, Kevin E.; Balázsi, Gábor; Gennaro, Maria Laura

    2016-01-01

    Accessory sigma factors, which reprogram RNA polymerase to transcribe specific gene sets, activate bacterial adaptive responses to noxious environments. Here we reconstruct the complete sigma factor regulatory network of the human pathogen Mycobacterium tuberculosis by an integrated approach. The approach combines identification of direct regulatory interactions between M. tuberculosis sigma factors in an E. coli model system, validation of selected links in M. tuberculosis, and extensive literature review. The resulting network comprises 41 direct interactions among all 13 sigma factors. Analysis of network topology reveals (i) a three-tiered hierarchy initiating at master regulators, (ii) high connectivity and (iii) distinct communities containing multiple sigma factors. These topological features are likely associated with multi-layer signal processing and specialized stress responses involving multiple sigma factors. Moreover, the identification of overrepresented network motifs, such as autoregulation and coregulation of sigma and anti-sigma factor pairs, provides structural information that is relevant for studies of network dynamics. PMID:27029515

  18. A sulfated metabolite produced by stf3 negatively regulates the virulence of Mycobacterium tuberculosis

    PubMed Central

    Mougous, Joseph D.; Senaratne, Ryan H.; Petzold, Christopher J.; Jain, Madhulika; Lee, Dong H.; Schelle, Michael W.; Leavell, Michael D.; Cox, Jeffery S.; Leary, Julie A.; Riley, Lee W.; Bertozzi, Carolyn R.

    2006-01-01

    Sulfated molecules have been shown to modulate isotypic interactions between cells of metazoans and heterotypic interactions between bacterial pathogens or symbionts and their eukaryotic host cells. Mycobacterium tuberculosis, the causative agent of tuberculosis, produces sulfated molecules that have eluded functional characterization for decades. We demonstrate here that a previously uncharacterized sulfated molecule, termed S881, is localized to the outer envelope of M. tuberculosis and negatively regulates the virulence of the organism in two mouse infection models. Furthermore, we show that the biosynthesis of S881 relies on the universal sulfate donor 3′-phosphoadenosine-5′-phosphosulfate and a previously uncharacterized sulfotransferase, stf3. These findings extend the known functions of sulfated molecules as general modulators of cell–cell interactions to include those between a bacterium and a human host. PMID:16537518

  19. MTBVAC vaccine is safe, immunogenic and confers protective efficacy against Mycobacterium tuberculosis in newborn mice

    PubMed Central

    Aguilo, Nacho; Uranga, Santiago; Marinova, Dessislava; Monzon, Marta; Badiola, Juan; Martin, Carlos

    2016-01-01

    Summary Development of novel more efficient preventive vaccines against tuberculosis (TB) is crucial to achieve TB eradication by 2050, one of the Millennium Development Goals (MDG) for the current century. MTBVAC is the first and only live attenuated vaccine based on a human isolate of Mycobacterium tuberculosis developed as BCG-replacement strategy in newborns that has entered first-in-human adult clinical trials. In this work, we characterize the safety, immunogenicity and protective efficacy of MTBVAC in a model of newborn C57/BL6 mice. Our data clearly indicate that MTBVAC is safe for newborn mice, and does not affect animal growth or organ development. In addition, MTBVAC-vaccinated mice at birth showed enhanced immunogenicity and better protection against M. tuberculosis challenge in comparison with BCG. PMID:26786657

  20. Crystallization and preliminary X-ray crystallographic studies of Mycobacterium tuberculosis chorismate mutase

    SciTech Connect

    Qamra, Rohini; Prakash, Prachee; Aruna, Bandi; Hasnain, Seyed E.; Mande, Shekhar C.

    2005-05-01

    Chorismate mutase from M. tuberculosis has been crystallized. Preliminary X-ray crystallographic studies reveal the occurrence of a dimeric molecule in the crystal asymmetric unit. Chorismate mutase catalyzes the first committed step in the biosynthesis of the aromatic amino acids phenylalanine and tyrosine in bacteria, fungi and higher plants. The recent re-annotation of the Mycobacterium tuberculosis genome has revealed the presence of a duplicate set of genes coding for chorismate mutase. The mycobacterial gene Rv1885c bears <20% sequence homology to other bacterial chorismate mutases, thus serving as a potential target for the development of inhibitors specific to the pathogen. The M. tuberculosis chorismate mutase was crystallized in space group C2 and the crystals diffracted to a resolution of 2.2 Å. Matthews coefficient and self-rotation function calculations revealed the presence of two monomers in the asymmetric unit.

  1. MTBVAC vaccine is safe, immunogenic and confers protective efficacy against Mycobacterium tuberculosis in newborn mice.

    PubMed

    Aguilo, Nacho; Uranga, Santiago; Marinova, Dessislava; Monzon, Marta; Badiola, Juan; Martin, Carlos

    2016-01-01

    Development of novel more efficient preventive vaccines against tuberculosis (TB) is crucial to achieve TB eradication by 2050, one of the Millennium Development Goals (MDG) for the current century. MTBVAC is the first and only live attenuated vaccine based on a human isolate of Mycobacterium tuberculosis developed as BCG-replacement strategy in newborns that has entered first-in-human adult clinical trials. In this work, we characterize the safety, immunogenicity and protective efficacy of MTBVAC in a model of newborn C57/BL6 mice. Our data clearly indicate that MTBVAC is safe for newborn mice, and does not affect animal growth or organ development. In addition, MTBVAC-vaccinated mice at birth showed enhanced immunogenicity and better protection against M. tuberculosis challenge in comparison with BCG. PMID:26786657

  2. Analysis of the role of Mycobacterium tuberculosis kasA gene mutations in isoniazid resistance.

    PubMed

    Sun, Y-J; Lee, A S G; Wong, S-Y; Paton, N I

    2007-08-01

    Previous studies have suggested that Mycobacterium tuberculosis kasA G312S and G269S gene mutations may represent sequence polymorphisms of the M. tuberculosis East-African-Indian (EAI) and T families, respectively, rather than relating to isoniazid resistance. The present study examined polymorphisms of these two codons in 98 drug-susceptible M. tuberculosis isolates (68 EAI and 30 T isolates). Twenty-eight isolates belonging to a sub-lineage of the EAI family had the kasA G312S mutation, but none of the 30 T isolates had the G269S mutation. The data suggest that the kasA G312S mutation is not related to isoniazid resistance, but represents a sequence polymorphism in a sub-lineage of the EAI family. PMID:17501974

  3. Disruption of the serine/threonine protein kinase H affects phthiocerol dimycocerosates synthesis in Mycobacterium tuberculosis

    PubMed Central

    Gómez-Velasco, Anaximandro; Bach, Horacio; Rana, Amrita K.; Cox, Liam R.; Bhatt, Apoorva; Besra, Gurdyal S.

    2013-01-01

    Mycobacterium tuberculosis possesses a complex cell wall that is unique and essential for interaction of the pathogen with its human host. Emerging evidence suggests that the biosynthesis of complex cell-wall lipids is mediated by serine/threonine protein kinases (STPKs). Herein, we show, using in vivo radiolabelling, MS and immunostaining analyses, that targeted deletion of one of the STPKs, pknH, attenuates the production of phthiocerol dimycocerosates (PDIMs), a major M. tuberculosis virulence lipid. Comparative protein expression analysis revealed that proteins in the PDIM biosynthetic pathway are differentially expressed in a deleted pknH strain. Furthermore, we analysed the composition of the major lipoglycans, lipoarabinomannan (LAM) and lipomannan (LM), and found a twofold higher LAM/LM ratio in the mutant strain. Thus, we provide experimental evidence that PknH contributes to the production and synthesis of M. tuberculosis cell-wall components. PMID:23412844

  4. Application of water-soluble nanofilters for collection of airborne Mycobacterium tuberculosis DNA in hospital wards.

    PubMed

    Vladimirsky, M A; Shipina, L K; Makeeva, E S; Alyapkina, Y S; Mikheev, A Y; Morozov, V N

    2016-05-01

    A simple inexpensive technique for collection of airborne biomarkers of nosocomial infections is described. Biomarkers were collected on water-soluble electrospun nanofilters attached to a household vacuum cleaner from 6-10m(3) of air in 10-15min within several wards of a tuberculosis clinic. Filters were then dissolved in water and tested for the presence of the IS6110 and regX3 genes of Mycobacterium tuberculosis (MTB) using real-time polymerase chain reaction. It was demonstrated that trace amounts of airborne MTB DNA (>3gene copies/m(3)) were always present in the air and on the surfaces in the wards occupied with tuberculosis patients having positive smear tests. PMID:27021397

  5. A functional role of Rv1738 in Mycobacterium tuberculosis persistence suggested by racemic protein crystallography.

    PubMed

    Bunker, Richard D; Mandal, Kalyaneswar; Bashiri, Ghader; Chaston, Jessica J; Pentelute, Bradley L; Lott, J Shaun; Kent, Stephen B H; Baker, Edward N

    2015-04-01

    Protein 3D structure can be a powerful predictor of function, but it often faces a critical roadblock at the crystallization step. Rv1738, a protein from Mycobacterium tuberculosis that is strongly implicated in the onset of nonreplicating persistence, and thereby latent tuberculosis, resisted extensive attempts at crystallization. Chemical synthesis of the L- and D-enantiomeric forms of Rv1738 enabled facile crystallization of the D/L-racemic mixture. The structure was solved by an ab initio approach that took advantage of the quantized phases characteristic of diffraction by centrosymmetric crystals. The structure, containing L- and D-dimers in a centrosymmetric space group, revealed unexpected homology with bacterial hibernation-promoting factors that bind to ribosomes and suppress translation. This suggests that the functional role of Rv1738 is to contribute to the shutdown of ribosomal protein synthesis during the onset of nonreplicating persistence of M. tuberculosis. PMID:25831534

  6. A functional role of Rv1738 in Mycobacterium tuberculosis persistence suggested by racemic protein crystallography

    PubMed Central

    Bunker, Richard D.; Mandal, Kalyaneswar; Bashiri, Ghader; Chaston, Jessica J.; Pentelute, Bradley L.; Lott, J. Shaun; Kent, Stephen B. H.; Baker, Edward N.

    2015-01-01

    Protein 3D structure can be a powerful predictor of function, but it often faces a critical roadblock at the crystallization step. Rv1738, a protein from Mycobacterium tuberculosis that is strongly implicated in the onset of nonreplicating persistence, and thereby latent tuberculosis, resisted extensive attempts at crystallization. Chemical synthesis of the l- and d-enantiomeric forms of Rv1738 enabled facile crystallization of the d/l-racemic mixture. The structure was solved by an ab initio approach that took advantage of the quantized phases characteristic of diffraction by centrosymmetric crystals. The structure, containing l- and d-dimers in a centrosymmetric space group, revealed unexpected homology with bacterial hibernation-promoting factors that bind to ribosomes and suppress translation. This suggests that the functional role of Rv1738 is to contribute to the shutdown of ribosomal protein synthesis during the onset of nonreplicating persistence of M. tuberculosis. PMID:25831534

  7. Synthesis and evaluation of a pyrazinoic acid prodrug in Mycobacterium tuberculosis

    PubMed Central

    Fernandes, João Paulo-dos Santos; Pavan, Fernando Rogerio; Leite, Clarice Queico Fujimura; Felli, Veni Maria Andres

    2013-01-01

    Tuberculosis (TB) is a disease caused mainly by infection of Mycobacterium tuberculosis affecting more than ten million people around the world. Despite TB can be treated, the rise of MDR-TB and XDR-TB cases put the disease in a worrying status. As pyrazinamide-resistant strains exhibit low or none pyrazinamidase activity, it is proposed that the active form of pyrazinamide (PZA) is pyrazinoic acid (POA), although this acid has poor penetration in mycobacteria. In this work, we present a convenient one-pot synthesis of 2-chloroethyl pyrazinoate, and its activity in M. tuberculosis H37Rv (ATCC27294) in MIC assay using the MABA technique. The obtained MIC of the compound was 3.96 g/mL, and discussion about the activity profile of some previously evaluated pyrazinoates is also performed. PMID:25161383

  8. Determination of in vitro susceptibility of Mycobacterium tuberculosis to cephalosporins by radiometric and conventional methods

    SciTech Connect

    Heifets, L.B.; Iseman, M.D.; Cook, J.L.; Lindholm-Levy, P.J.; Drupa, I.

    1985-01-01

    Among eight cephalosporins and cephamycins tested in preliminary in vitro screening against Mycobacterium tuberculosis, the most promising for further study was found to be ceforanide, followed by ceftizoxime, cephapirin, and cefotaxime. Moxalactam, cefoxitin, cefamandole, and cephalothin were found to be not active enough against M. tuberculosis to be considered for further in vitro studies. The antibacterial activity of various ceforanide concentrations was investigated by three methods: (i) the dynamics of radiometric readings (growth index) in 7H12 broth; (ii) the number of CFU in the same medium; and (iii) the proportion method on 7H11 agar plates. There was a good correlation among the results obtained with these methods. The MIC for most strains ranged from 6.0 to 25.0 micrograms/ml. The BACTEC radiometric method is a reliable, rapid, and convenient method for preliminary screening and determination of the level of antibacterial activity of drugs not commonly used against M. tuberculosis.

  9. Latent Mycobacterium tuberculosis Infection in Liver Transplant Recipients—Controversies in Current Diagnosis and Management

    PubMed Central

    Rajagopala, Srinivas; Olithselvan, A; Varghese, Joy; Shanmugam, Naresh; Rela, Mohamed

    2011-01-01

    Liver transplantation for end-stage liver disease is increasingly being undertaken in India.1 Routine tuberculin skin testing (TST) for latent Mycobacterium tuberculosis infection (LTBI) and isoniazid prophylaxis in TST-positive liver-transplant recipients (LTRs) is recommended2,3 but seldom implemented worldwide.4–7 The role of TST-testing and isoniazid prophylaxis in LTRs remains further undefined in high prevalence areas, including India. We describe the burden of LTBI in LTRs; the epidemiological aspects of M. tuberculosis infection in high prevalence areas; identifiable risk factors for M. tuberculosis infection; the limitations of current diagnostic techniques for LTBI in LTRs and the efficacy and toxicity of isoniazid prophylaxis in TST-positive LTRs and suggest directions for future investigations in this area. PMID:25755308

  10. Tuberculosis in Elephants: Antibody Responses to Defined Antigens of Mycobacterium tuberculosis, Potential for Early Diagnosis, and Monitoring of Treatment

    PubMed Central

    Lyashchenko, Konstantin P.; Greenwald, Rena; Esfandiari, Javan; Olsen, John H.; Ball, Ray; Dumonceaux, Genevieve; Dunker, Freeland; Buckley, Carol; Richard, Michael; Murray, Suzan; Payeur, Janet B.; Andersen, Peter; Pollock, John M.; Mikota, Susan; Miller, Michele; Sofranko, Denise; Waters, W. Ray

    2006-01-01

    Tuberculosis (TB) in elephants is a re-emerging zoonotic disease caused primarily by Mycobacterium tuberculosis. Current diagnosis relies on trunk wash culture, the only officially recognized test, which has serious limitations. Innovative and efficient diagnostic methods are urgently needed. Rapid identification of infected animals is a crucial prerequisite for more effective control of TB, as early diagnosis allows timely initiation of chemotherapy. Serology has diagnostic potential, although key antigens have not been identified and optimal immunoassay formats are not established. To characterize the humoral responses in elephant TB, we tested 143 serum samples collected from 15 elephants over time. These included 48 samples from five culture-confirmed TB cases, of which four were in Asian elephants infected with M. tuberculosis and one was in an African elephant with Mycobacterium bovis. Multiantigen print immunoassay (MAPIA) employing a panel of 12 defined antigens was used to identify serologic correlates of active disease. ESAT-6 was the immunodominant antigen recognized in elephant TB. Serum immunoglobulin G antibodies to ESAT-6 and other proteins were detected up to 3.5 years prior to culture of M. tuberculosis from trunk washes. Antibody levels to certain antigens gradually decreased in response to antitubercular therapy, suggesting the possibility of treatment monitoring. In addition to MAPIA, serum samples were evaluated with a recently developed rapid test (RT) based on lateral flow technology (ElephantTB STAT-PAK). Similarly to MAPIA, infected elephants were identified using the RT up to 4 years prior to positive culture. These findings demonstrate the potential for TB surveillance and treatment monitoring using the RT and MAPIA, respectively. PMID:16829608

  11. Evaluation of large genotypic Mycobacterium tuberculosis clusters: contributions from remote and recent transmission.

    PubMed

    Teeter, Larry D; Ha, Ngan P; Ma, Xin; Wenger, Jane; Cronin, Wendy A; Musser, James M; Graviss, Edward A

    2013-12-01

    Tuberculosis genotypic clustering is used as a proxy for recent transmission. The association between clustering and recent transmission becomes problematic when the genotyping method lacks specificity in defining a cluster, as well as for clusters with extensive jurisdictional histories and/or common genotypes. We investigated the four largest spoligotype/12 loci MIRU-VNTR-defined clusters in Harris County, Texas from 2006-2012 to determine their historical contribution to tuberculosis morbidity, estimate the contributions from recent and remote transmission, and determine the impact of secondary genotyping on cluster definition. The clusters contained 189, 64, 51 and 38 cases. Each cluster was linked to cluster(s) previously identified by Houston Tuberculosis Initiative; 3 since 1995 and the fourth in 2002. Among cases for which timing of Mycobacterium tuberculosis transmission relative to tuberculosis disease could be ascertained, nearly equal proportions were associated with recent and remote transmission. The extent to which genotyping with an additional 12 MIRU-VNTR loci modified the cluster definition varied from little or no impact for the two smaller clusters to moderate impact for the larger clusters. Tuberculosis control measures to reduce morbidity associated with large clusters must involve strategies to identify and treat individuals who recently acquired infection, as well as persons infected for years. PMID:24388648

  12. Effects of genetic variability of Mycobacterium tuberculosis strains on the presentation of disease.

    PubMed

    Malik, Aeesha N J; Godfrey-Faussett, Peter

    2005-03-01

    The nature of the variability in the clinical and epidemiological consequences of Mycobacterium tuberculosis infection remains poorly understood. Environmental and host factors that contribute to the outcome of infection and disease presentation are well recognised, but the role of bacterial factors has been more elusive. The rapid increase in the understanding of the molecular basis of M tuberculosis over the past decades has revived research into its pathogenesis. DNA fingerprinting techniques have been used to distinguish between strains of M tuberculosis, and efforts to characterise the strains present within populations have led to increased understanding of their global distribution. This research has shown that in certain areas a small number of strains are causing a disproportionate number of cases of the disease. The sequencing of the complete genome of M tuberculosis has accelerated the development of molecular techniques to differentiate strains according to their genetic polymorphisms. Investigation into the reasons why some strains are predominant by genetic strain-typing techniques may clarify which bacterial factors contribute to disease. This knowledge has the potential to influence control and prevention strategies for tuberculosis in the future. However, there are still limitations in these techniques and their results. This review discusses molecular epidemiology and genetic studies, and their contribution to the understanding of the links between genotypic and phenotypic characteristics of M tuberculosis strains. PMID:15766652

  13. Rv3351c, a Mycobacterium tuberculosis gene that affects bacterial growth and alveolar epithelial cell viability.

    PubMed

    Pavlicek, Rebecca L; Fine-Coulson, Kari; Gupta, Tuhina; Quinn, Frederick D; Posey, James E; Willby, Melisa; Castro-Garza, Jorge; Karls, Russell K

    2015-12-01

    Despite the interactions known to occur between various lower respiratory tract pathogens and alveolar epithelial cells (AECs), few reports examine factors influencing the interplay between Mycobacterium tuberculosis bacilli and AECs during infection. Importantly, in vitro studies have demonstrated that the M. tuberculosis hbha and esxA gene products HBHA and ESAT6 directly or indirectly influence AEC survival. In this report, we identify Rv3351c as another M. tuberculosis gene that impacts the fate of both the pathogen and AEC host. Intracellular replication of an Rv3351c mutant in the human AEC type II pneumocyte cell line A549 was markedly reduced relative to the complemented mutant and parent strain. Deletion of Rv3351c diminished the release of lactate dehydrogenase and decreased uptake of trypan blue vital stain by host cells infected with M. tuberculosis bacilli, suggesting attenuated cytotoxic effects. Interestingly, an isogenic hbha mutant displayed reductions in AEC killing similar to those observed for the Rv3351c mutant. This opens the possibility that multiple M. tuberculosis gene products interact with AECs. We also observed that Rv3351c aids intracellular replication and survival of M. tuberculosis in macrophages. This places Rv3351c in the same standing as HBHA and ESAT6, which are important factors in AECs and macrophages. Defining the mechanism(s) by which Rv3351c functions to aid pathogen survival within the host may lead to new drug or vaccine targets. PMID:26492080

  14. Modulation of host ubiquitin system genes in human endometrial cell line infected with Mycobacterium tuberculosis.

    PubMed

    Meenu, S; Thiagarajan, S; Ramalingam, Sudha; Michael, A; Ramalingam, Sankaran

    2016-04-01

    Endometrium is one of the most commonly affected sites in genital tuberculosis. The understanding of its interaction with the tubercle bacilli is of paramount importance for studying the pathogenesis of this disease. The main objective of this work was to study the interplay between Mycobacterium tuberculosis and host endometrial epithelial cell lines (Ishikawa cell lines), and to identify the differentially expressed genes upon tuberculosis infection. To study this, suppression subtractive hybridization library was constructed using M. tuberculosis H37Rv-infected Ishikawa cell line harvested 24 h post-infection. The subtracted cDNA library was screened, and 105 differentially expressed genes were identified and grouped based on their functions. Since ubiquitination process has gained importance in targeting M. tuberculosis to xenophagy, ubiquitin system genes obtained in the library were selected, and time course analysis of their gene expression was performed. We observed an upregulation of mkrn1 and cops5 and downregulation of zfp91, ndfip2, ube2f, rnft1, psmb6, and psmd13 at 24 h post-infection. From the results obtained, we surmise that ubiquitination pathway genes may have roles in combating tuberculosis which are yet uncharted. PMID:26403675

  15. A 38-kD Mycobacterium tuberculosis antigen associated with infection. Its isolation and serologic evaluation.

    PubMed Central

    Espitia, C; Cervera, I; González, R; Mancilla, R

    1989-01-01

    To identify antigens that could be specifically associated with tuberculosis infection, the antibody response to Mycobacterium tuberculosis antigens of patients with pulmonary tuberculosis and of healthy individuals were compared by immunoblot. In healthy individuals, serum antibodies were found in the majority of cases. Bands of 60 and 32-31 kilodaltons (kD) were the antigens more frequently recognized by antibodies of normal sera (55.8 and 64.7%, respectively). In patients with pulmonary tuberculosis, the number and intensity of the developed antigen bands were much higher than in normal individuals. Antigens reacting preferentially with tuberculosis sera were also identified. Furthermore, a unique disease-associated protein antigen of 38 kD was found to react with 57% of patients' sera but with none of the controls. This antigen was isolated by elution from nitrocellulose membranes and tested as an ELISA reagent in the serodiagnosis of pulmonary tuberculosis. A specificity of 0.96 and sensitivity of 0.68 were obtained. Images Fig. 1 Fig. 3 PMID:2478322

  16. Bioinformatics Identification of Drug Resistance-Associated Gene Pairs in Mycobacterium tuberculosis.

    PubMed

    Cui, Ze-Jia; Yang, Qing-Yong; Zhang, Hong-Yu; Zhu, Qiang; Zhang, Qing-Ye

    2016-01-01

    Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis (Mtb). Due to the extensive use of anti-tuberculosis drugs and the development of mutations, the emergence and spread of multidrug-resistant tuberculosis is recognized as one of the most dangerous threats to global tuberculosis control. Some single mutations have been identified to be significantly linked with drug resistance. However, the prior research did not take gene-gene interactions into account, and the emergence of transmissible drug resistance is connected with multiple genetic mutations. In this study we use the bioinformatics software GBOOST (The Hong Kong University, Clear Water Bay, Kowloon, Hong Kong, China) to calculate the interactions of Single Nucleotide Polymorphism (SNP) pairs and identify gene pairs associated with drug resistance. A large part of the non-synonymous mutations in the drug target genes that were included in the screened gene pairs were confirmed by previous reports, which lent sound solid credits to the effectiveness of our method. Notably, most of the identified gene pairs containing drug targets also comprise Pro-Pro-Glu (PPE) family proteins, suggesting that PPE family proteins play important roles in the drug resistance of Mtb. Therefore, this study provides deeper insights into the mechanisms underlying anti-tuberculosis drug resistance, and the present method is useful for exploring the drug resistance mechanisms for other microorganisms. PMID:27618895

  17. Mycobacterium tuberculosis Cluster with Developing Drug Resistance, New York, New York, USA, 2003–2009

    PubMed Central

    Proops, Douglas; Moonan, Patrick K.; Munsiff, Sonal S.; Kreiswirth, Barry N.; Kurepina, Natalia; Goranson, Christopher; Ahuja, Shama D.

    2011-01-01

    In 2004, identification of patients infected with the same Mycobacterium tuberculosis strain in New York, New York, USA, resulted in an outbreak investigation. The investigation involved data collection and analysis, establishing links between patients, and forming transmission hypotheses. Fifty-four geographically clustered cases were identified during 2003–2009. Initially, the M. tuberculosis strain was drug susceptible. However, in 2006, isoniazid resistance emerged, resulting in isoniazid-resistant M. tuberculosis among 17 (31%) patients. Compared with patients with drug-susceptible M. tuberculosis, a greater proportion of patients with isoniazid-resistant M. tuberculosis were US born and had a history of illegal drug use. No patients named one another as contacts. We used patient photographs to identify links between patients. Three links were associated with drug use among patients infected with isoniazid-resistant M. tuberculosis. The photographic method would have been more successful if used earlier in the investigation. Name-based contact investigation might not identify all contacts, particularly when illegal drug use is involved. PMID:21392426

  18. Vaccination with an Attenuated Ferritin Mutant Protects Mice against Virulent Mycobacterium tuberculosis

    PubMed Central

    Subbian, Selvakumar; Pandey, Ruchi; Soteropoulos, Patricia; Rodriguez, G. Marcela

    2015-01-01

    Mycobacterium tuberculosis the causative agent of tuberculosis affects millions of people worldwide. New tools for treatment and prevention of tuberculosis are urgently needed. We previously showed that a ferritin (bfrB) mutant of M. tuberculosis has altered iron homeostasis and increased sensitivity to antibiotics and to microbicidal effectors produced by activated macrophages. Most importantly, M. tuberculosis lacking BfrB is strongly attenuated in mice, especially, during the chronic phase of infection. In this study, we examined whether immunization with a bfrB mutant could confer protection against subsequent infection with virulent M. tuberculosis in a mouse model. The results show that the protection elicited by immunization with the bfrB mutant is comparable to BCG vaccination with respect to reduction of bacterial burden. However, significant distinctions in the disease pathology and host genome-wide lung transcriptome suggest improved containment of Mtb infection in animals vaccinated with the bfrB mutant, compared to BCG. We found that downmodulation of inflammatory response and enhanced fibrosis, compared to BCG vaccination, is associated with the protective response elicited by the bfrB mutant. PMID:26339659

  19. Phosphoproteomics analysis of a clinical Mycobacterium tuberculosis Beijing isolate: expanding the mycobacterial phosphoproteome catalog

    PubMed Central

    Fortuin, Suereta; Tomazella, Gisele G.; Nagaraj, Nagarjuna; Sampson, Samantha L.; Gey van Pittius, Nicolaas C.; Soares, Nelson C.; Wiker, Harald G.; de Souza, Gustavo A.; Warren, Robin M.

    2015-01-01

    Reversible protein phosphorylation, regulated by protein kinases and phosphatases, mediates a switch between protein activity and cellular pathways that contribute to a large number of cellular processes. The Mycobacterium tuberculosis genome encodes 11 Serine/Threonine kinases (STPKs) which show close homology to eukaryotic kinases. This study aimed to elucidate the phosphoproteomic landscape of a clinical isolate of M. tuberculosis. We performed a high throughput mass spectrometric analysis of proteins extracted from an early-logarithmic phase culture. Whole cell lysate proteins were processed using the filter-aided sample preparation method, followed by phosphopeptide enrichment of tryptic peptides by strong cation exchange (SCX) and Titanium dioxide (TiO2) chromatography. The MaxQuant quantitative proteomics software package was used for protein identification. Our analysis identified 414 serine/threonine/tyrosine phosphorylated sites, with a distribution of S/T/Y sites; 38% on serine, 59% on threonine and 3% on tyrosine; present on 303 unique peptides mapping to 214 M. tuberculosis proteins. Only 45 of the S/T/Y phosphorylated proteins identified in our study had been previously described in the laboratory strain H37Rv, confirming previous reports. The remaining 169 phosphorylated proteins were newly identified in this clinical M. tuberculosis Beijing strain. We identified 5 novel tyrosine phosphorylated proteins. These findings not only expand upon our current understanding of the protein phosphorylation network in clinical M. tuberculosis but the data set also further extends and complements previous knowledge regarding phosphorylated peptides and phosphorylation sites in M. tuberculosis. PMID:25713560

  20. Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology.

    PubMed Central

    Kamerbeek, J; Schouls, L; Kolk, A; van Agterveld, M; van Soolingen, D; Kuijper, S; Bunschoten, A; Molhuizen, H; Shaw, R; Goyal, M; van Embden, J

    1997-01-01

    Widespread use of DNA restriction fragment length polymorphism (RFLP) to differentiate strains of Mycobacterium tuberculosis to monitor the transmission of tuberculosis has been hampered by the need to culture this slow-growing organism and by the level of technical sophistication needed for RFLP typing. We have developed a simple method which allows simultaneous detection and typing of M. tuberculosis in clinical specimens and reduces the time between suspicion of the disease and typing from 1 or several months to 1 or 3 days. The method is based on polymorphism of the chromosomal DR locus, which contains a variable number of short direct repeats interspersed with nonrepetitive spacers. The method is referred to as spacer oligotyping or "spoligotyping" because it is based on strain-dependent hybridization patterns of in vitro-amplified DNA with multiple spacer oligonucleotides. Most of the clinical isolates tested showed unique hybridization patterns, whereas outbreak strains shared the same spoligotype. The types obtained from direct examination of clinical samples were identical to those obtained by using DNA from cultured M. tuberculosis. This novel preliminary study shows that the novel method may be a useful tool for rapid disclosure of linked outbreak cases in a community, in hospitals, or in other institutions and for monitoring of transmission of multidrug-resistant M. tuberculosis. Unexpectedly, spoligotyping was found to differentiate M. bovis from M. tuberculosis, a distinction which is often difficult to make by traditional methods. PMID:9157152

  1. Subcellular Localization of the Intracellular Survival-Enhancing Eis Protein of Mycobacterium tuberculosis

    PubMed Central

    Dahl, John L.; Wei, Jun; Moulder, James W.; Laal, Suman; Friedman, Richard L.

    2001-01-01

    Mycobacterium tuberculosis is a facultative intracellular pathogen that has evolved the ability to survive and multiply within human macrophages. It is not clear how M. tuberculosis avoids the destructive action of macrophages, but this ability is fundamental in the pathogenicity of tuberculosis. A gene previously identified in M. tuberculosis, designated eis, was found to enhance intracellular survival of Mycobacterium smegmatis in the human macrophage-like cell line U-937 (J. Wei et al., J. Bacteriol. 182:377–384, 2000). When eis was introduced into M. smegmatis on a multicopy vector, sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the appearance of a unique 42-kDa protein band corresponding to the predicted molecular weight of the eis gene product. This band was electroeluted from the gel with a purity of >90% and subjected to N-terminal amino acid sequencing, which demonstrated that the 42-kDa band was indeed the protein product of eis. The Eis protein produced by M. tuberculosis H37Ra had an identical N-terminal amino acid sequence. A synthetic polypeptide corresponding to a carboxyl-terminal region of the deduced eis protein sequence was used to generate affinity-purified rabbit polyclonal antibodies that reacted with the 42-kDa protein in Western blot analysis. Hydropathy profile analysis showed the Eis protein to be predominantly hydrophilic with a potential hydrophobic amino terminus. Phase separation of M. tuberculosis H37Ra lysates by the nonionic detergent Triton X-114 revealed the Eis protein in both the aqueous and detergent phases. After fractionation of M. tuberculosis by differential centrifugation, Eis protein appeared mainly in the cytoplasmic fraction but also in the membrane, cell wall, and culture supernatant fractions as well. Forty percent of the sera from pulmonary tuberculosis patients tested for anti-Eis antibody gave positive reactions in Western blot analysis. Although the function of Eis remains unknown, evidence

  2. Multi-Stage Tuberculosis Subunit Vaccine Candidate LT69 Provides High Protection against Mycobacterium tuberculosis Infection in Mice

    PubMed Central

    Niu, Hongxia; Peng, Jinxiu; Bai, Chunxiang; Liu, Xun; Hu, Lina; Luo, Yanping; Wang, Bingxiang; Zhang, Ying; Chen, Jianzhu; Yu, Hongjuan; Xian, Qiaoyang; Zhu, Bingdong

    2015-01-01

    Effective tuberculosis (TB) vaccine should target tubercle bacilli with various metabolic states and confer long-term protective immunity. In this study, we constructed a novel multi-stage TB subunit vaccine based on fusion protein ESAT6-Ag85B-MPT64(190-198)-Mtb8.4-HspX (LT69 for short) which combined early expressed antigens and latency-associated antigen. The fusion protein was mixed with an adjuvant being composed of N, N’-dimethyl-N, N’-dioctadecylammonium bromide (DDA) and polyriboinosinic polyribocytidylic acid (PolyI:C) to construct subunit vaccine, whose immunogenicity and protective ability were evaluated in C57BL/6 mice. The results showed that LT69 had strong immunogenicity and high protective effect against Mycobacterium tuberculosis (M. tuberculosis) H37Rv aerosol challenge. Low-dose (2 μg) of LT69 generated long-term immune memory responses and provided effective protection, which was even higher than traditional vaccine BCG did at 30 weeks post the last vaccination. In conclusion, multistage subunit vaccine LT69 showed high and long-term protection against M. tuberculosis infection in mice, whose effect could be enhanced by using a relative low dosage of antigen. PMID:26098302

  3. Evaluation of method for secondary DNA typing of Mycobacterium tuberculosis with pTBN12 in epidemiologic study of tuberculosis.

    PubMed Central

    Yang, Z; Chaves, F; Barnes, P F; Burman, W J; Koehler, J; Eisenach, K D; Bates, J H; Cave, M D

    1996-01-01

    Secondary fingerprinting of Mycobacterium tuberculosis DNA with a probe containing the polymorphic GC-rich repetitive sequence present in pTBN12 has been found to have greater discriminating power than does fingerprinting with the insertion sequence IS6110 for strains carrying few copies of IS6110. To validate the use of pTBN12 fingerprinting in the molecular epidemiology of tuberculosis, M. tuberculosis isolates from 67 patients in five states in the United States and in Spain were fingerprinted with both IS6110 and pTBN12. Epidemiologic links among the 67 patients were evaluated by patient interview and/or review of medical records. The 67 isolates had 5 IS6110 fingerprint patterns with two to five copies of IS6110 and 18 pTBN12 patterns, of which 10 were shared by more than 1 isolate. Epidemiologic links are consistently found among patients whose isolates had identical pTBN12 patterns, whereas no links were found among patients whose isolates had unique pTBN12 patterns. This suggests that pTBN12 fingerprinting is a useful tool to identify epidemiologically linked tuberculosis patients whose isolates have identical IS6110 fingerprints containing fewer than six fragments. PMID:8940446

  4. A study on pre-XDR & XDR tuberculosis & their prevalent genotypes in clinical isolates of Mycobacterium tuberculosis in north India

    PubMed Central

    Singhal, Parul; Dixit, Pratima; Singh, Pooja; Jaiswal, Indu; Singh, Mastan; Jain, Amita

    2016-01-01

    Background & objectives: Pre-extensively drug resistant (pre-XDR) and extensively drug resistant tuberculosis (XDR-TB) have been areas of growing concern, and are posing threat to global efforts of TB control. The present study was planned to study the presence of pre-XDR and XDR Mycobacterium tuberculosis and their genotypes in clinical isolates obtained from previously treated cases of pulmonary TB. Methods: A total of 219 isolates obtained from previously treated cases of pulmonary TB were subjected to first-line (streptomycin, isoniazid, rifampicin and ethambutol) and second-line (ofloxacin, kanamycin, capreomycin and amikacin) drug susceptibility testing on solid Lowenstein-Jensen medium by proportion method. Genotyping was done for pre-XDR and XDR-TB isolates using 12 loci Mycobacterial Interspersed Repetitive Units-Variable Number Tandem Repeats (MIRU-VNTR). Results: Multi-drug resistance was observed in 39.7 per cent (87/219) isolates. Pre-XDR and XDR M. tuberculosis isolates amongst 87 multi-drug resistant (MDR) TB isolates were 43 (49.4%) and 10 (11.4%), respectively. Two most dominant genotypes among pre-XDR and XDR M. tuberculosis isolates were Beijing and Delhi/CAS types. Interpretation & conclusions: Resistance to second-line anti-tubercular drugs should be routinely assessed in areas endemic for TB. Similar genotype patterns were seen in pre-XDR and XDR-TB isolates. Beijing and Delhi/CAS were predominant genotypes. PMID:27241648

  5. Genetic Diversity of Mycobacterium tuberculosis Complex Isolated from Tuberculosis Patients in Bahir Dar City and Its Surroundings, Northwest Ethiopia.

    PubMed

    Nuru, Anwar; Mamo, Gezahegne; Worku, Adane; Admasu, Aschalew; Medhin, Girmay; Pieper, Rembert; Ameni, Gobena

    2015-01-01

    The knowledge of the diversity of strains of Mycobacterium tuberculosis complex (MTBC) species in a specific geographical region can contribute to the control of tuberculosis (TB). This study was conducted to identify the MTBC isolates to the species and spoligotype international type (SIT) level by spoligotyping. A total of 168 MTBC isolates were recovered from TB patients, spoligotyped, and their patterns were compared with those of the strains registered in the SITVIT2 database. Of 168 isolates spoligotyped, 89 patterns were identified. Ninety-eight isolates were clustered into 19 strain groups with clustering percentage of 58.3%. Forty-four strains matched the preexisting SITs in the SITVIT2 database. The dominant strains were SIT289, SIT134, and SIT3411, comprising 16.7% (28/168), 7.14% (12/168), and 4.76% (8/168) of the isolates, respectively. Euro-American (51.2%), East-African-Indian (34.5%), and M. africanum (9.52%) were the major lineages identified. Two strains of M. bovis were isolated from TB lymphadenitis cases. The high percentage of clustered strains of M. tuberculosis could suggest that a small number of lineages of M. tuberculosis are causing the disease in the area while isolation of M. bovis could suggest its zoonotic potential. Additionally, identification of M. africanum requires further confirmation by tools with a better discriminatory power. PMID:26491657

  6. Genetic Diversity of Mycobacterium tuberculosis Complex Isolated from Tuberculosis Patients in Bahir Dar City and Its Surroundings, Northwest Ethiopia

    PubMed Central

    Nuru, Anwar; Mamo, Gezahegne; Worku, Adane; Admasu, Aschalew; Medhin, Girmay; Pieper, Rembert; Ameni, Gobena

    2015-01-01

    The knowledge of the diversity of strains of Mycobacterium tuberculosis complex (MTBC) species in a specific geographical region can contribute to the control of tuberculosis (TB). This study was conducted to identify the MTBC isolates to the species and spoligotype international type (SIT) level by spoligotyping. A total of 168 MTBC isolates were recovered from TB patients, spoligotyped, and their patterns were compared with those of the strains registered in the SITVIT2 database. Of 168 isolates spoligotyped, 89 patterns were identified. Ninety-eight isolates were clustered into 19 strain groups with clustering percentage of 58.3%. Forty-four strains matched the preexisting SITs in the SITVIT2 database. The dominant strains were SIT289, SIT134, and SIT3411, comprising 16.7% (28/168), 7.14% (12/168), and 4.76% (8/168) of the isolates, respectively. Euro-American (51.2%), East-African-Indian (34.5%), and M. africanum (9.52%) were the major lineages identified. Two strains of M. bovis were isolated from TB lymphadenitis cases. The high percentage of clustered strains of M. tuberculosis could suggest that a small number of lineages of M. tuberculosis are causing the disease in the area while isolation of M. bovis could suggest its zoonotic potential. Additionally, identification of M. africanum requires further confirmation by tools with a better discriminatory power. PMID:26491657

  7. Dxr is essential in Mycobacterium tuberculosis and fosmidomycin resistance is due to a lack of uptake

    PubMed Central

    Brown, Amanda C; Parish, Tanya

    2008-01-01

    Fosmidomycin is a phosphonic antibiotic which inhibits 1-deoxy-D-xylulose 5-phosphate reductoisomerase (Dxr), the first committed step of the non-mevalonate pathway of isoprenoid biosynthesis. In Mycobacterium tuberculosis Dxr is encoded by Rv2870c, and although the antibiotic has been shown to inhibit the recombinant enzyme [1], mycobacteria are intrinsically resistant to fosmidomycin at the whole cell level. Fosmidomycin is a hydrophilic molecule and in many bacteria its uptake is an active process involving a cAMP dependent glycerol-3-phosphate transporter (GlpT). The fact that there is no glpT homologue in the M. tuberculosis genome and the highly impervious nature of the hydrophobic mycobacterial cell wall suggests that resistance may be due to a lack of cellular penetration. Results We demonstrated that dxr (Rv2780c) is an essential gene in M. tuberculosis, since we could not delete the chromosomal copy unless a second functional copy was provided on an integrating vector. This confirmed that the intracellular target of fosmidomycin was essential as well as sensitive. We looked at the uptake of fosmidomycin in two mycobacterial species, the slow-growing pathogenic M. tuberculosis and the fast-growing, saprophytic Mycobacterium smegmatis; both species were resistant to fosmidomycin to a high level. Fosmidomycin was not accumulated intra-cellularly in M. tuberculosis or M. smegmatis but remained in the extra-cellular medium. In contrast, fosmidomycin uptake was confirmed in the sensitive organism, Escherichia coli. We established that the lack of intra-cellular accumulation was not due to efflux, since efflux pump inhibitors had no effect on fosmidomycin resistance. Finally, we demonstrated that fosmidomycin was not modified by mycobacterial cells or by extracts but remained in a fully functional state. Conclusion Taken together, these data demonstrate that fosmidomycin resistance in M. tuberculosis and M. smegmatis results from a lack of penetration of the

  8. The Mycobacterium tuberculosis Outer Membrane Channel Protein CpnT Confers Susceptibility to Toxic Molecules

    PubMed Central

    Danilchanka, Olga; Pires, David

    2015-01-01

    Mycobacterium tuberculosis, the causative agent of tuberculosis, is protected from toxic solutes by an effective outer membrane permeability barrier. Recently, we showed that the outer membrane channel protein CpnT is required for efficient nutrient uptake by M. tuberculosis and Mycobacterium bovis BCG. In this study, we found that the cpnT mutant of M. bovis BCG is more resistant than the wild type to a large number of drugs and antibiotics, including rifampin, ethambutol, clarithromycin, tetracycline, and ampicillin, by 8- to 32-fold. Furthermore, the cpnT mutant of M. bovis BCG was 100-fold more resistant to nitric oxide, a major bactericidal agent required to control M. tuberculosis infections in mice. Thus, CpnT constitutes the first outer membrane susceptibility factor in slow-growing mycobacteria. The dual functions of CpnT in uptake of nutrients and mediating susceptibility to toxic molecules are reflected in macrophage infection experiments: while loss of CpnT was detrimental for M. bovis BCG in macrophages that enable bacterial replication, presumably due to inadequate nutrient uptake, it conferred a survival advantage in macrophages that mount a strong bactericidal response. Importantly, the cpnT gene showed a significantly higher density of nonsynonymous mutations in drug-resistant clinical M. tuberculosis strains, indicating that CpnT is under selective pressure in human tuberculosis and/or during chemotherapy. Our results indicate that the CpnT channel constitutes an outer membrane gateway controlling the influx of nutrients and toxic molecules into slow-growing mycobacteria. This study revealed that reducing protein-mediated outer membrane permeability might constitute a new drug resistance mechanism in slow-growing mycobacteria. PMID:25645841

  9. Analytical and Clinical Evaluation of the Epistem Genedrive Assay for Detection of Mycobacterium tuberculosis.

    PubMed

    Shenai, Shubhada; Armstrong, Derek T; Valli, Eloise; Dolinger, David L; Nakiyingi, Lydia; Dietze, Reynaldo; Dalcolmo, Margareth Pretti; Nicol, Mark P; Zemanay, Widaad; Manabe, Yuka; Hadad, David Jamil; Marques-Rodrigues, Patricia; Palaci, Moises; Peres, Renata L; Gaeddert, Mary; Armakovitch, Sandra; Nonyane, Bareng A S; Denkinger, Claudia M; Banada, Padmapriya; Joloba, Moses L; Ellner, Jerrold; Boehme, Catharina; Alland, David; Dorman, Susan E

    2016-04-01

    The Epistem Genedrive assay rapidly detects the Mycobacterium tuberculosis omplex from sputum and is currently available for clinical use. However, the analytical and clinical performance of this test has not been fully evaluated. The analytical limit of detection (LOD) of the Genedrive PCR amplification was tested with genomic DNA; the performance of the complete (sample processing plus amplification) system was tested by spiking M. tuberculosismc(2)6030 cells into distilled water andM. tuberculosis-negative sputum. Specificity was tested using common respiratory pathogens and nontuberculosis mycobacteria. A clinical evaluation enrolled adults with suspected pulmonary tuberculosis, obtained three sputum samples from each participant, and compared the accuracy of the Gene drive to that of the Xpert MTB/RIF assay using M. tuberculosiscultures as the reference standard. The Genedrive assay had an LOD of 1 pg/μl (100 genomic DNA copies/reaction). The LODs of the system were 2.5 × 10(4)CFU/ml and 2.5 × 10(5)CFU/ml for cells spiked into water and sputum, respectively. False-positiverpoBprobe signals were observed in 3/32 (9.4%) of the negative controls and also in few samples containing Mycobacterium abscessus,Mycobacterium gordonae, o rMycobacterium thermoresistibile In the clinical study, among 336 analyzed participants, the overall sensitivities for the tuberculosis case detection of Gene drive, Xpert, and smear microscopy were 45.4% (95% confidence interval [CI], 35.2% to 55.8%), 91.8% (95% CI, 84.4% to 96.4%), and 77.3% (95% CI, 67.7% to 85.2%), respectively. The sensitivities of Gene drive and Xpert for the detection of smear-microscopy-negative tuberculosis were 0% (95% CI, 0% to 15.4%) and 68.2% (95% CI, 45.1% to 86.1%), respectively. The Genedrive assay did not meet performance standards recommended by the World Health Organization for a smear microscopy replacement tuberculosis test. Epistem is working on modifications to improve the assay. PMID:26865685

  10. LL-37 immunomodulatory activity during Mycobacterium tuberculosis infection in macrophages.

    PubMed

    Torres-Juarez, Flor; Cardenas-Vargas, Albertina; Montoya-Rosales, Alejandra; González-Curiel, Irma; Garcia-Hernandez, Mariana H; Enciso-Moreno, Jose A; Hancock, Robert E W; Rivas-Santiago, Bruno

    2015-12-01

    Tuberculosis is one of the most important infectious diseases worldwide. The susceptibility to this disease depends to a great extent on the innate immune response against mycobacteria. Host defense peptides (HDP) are one of the first barriers to counteract infection. Cathelicidin (LL-37) is an HDP that has many immunomodulatory effects besides its weak antimicrobial activity. Despite advances in the study of the innate immune response in tuberculosis, the immunological role of LL-37 during M. tuberculosis infection has not been clarified. Monocyte-derived macrophages were infected with M. tuberculosis strain H37Rv and then treated with 1, 5, or 15 μg/ml of exogenous LL-37 for 4, 8, and 24 h. Exogenous LL-37 decreased tumor necrosis factor alpha (TNF-α) and interleukin-17 (IL-17) while inducing anti-inflammatory IL-10 and transforming growth factor β (TGF-β) production. Interestingly, the decreased production of anti-inflammatory cytokines did not reduce antimycobacterial activity. These results are consistent with the concept that LL-37 can modulate the expression of cytokines during mycobacterial infection and this activity was independent of the P2X7 receptor. Thus, LL-37 modulates the response of macrophages during infection, controlling the expression of proinflammatory and anti-inflammatory cytokines. PMID:26351280

  11. LL-37 Immunomodulatory Activity during Mycobacterium tuberculosis Infection in Macrophages

    PubMed Central

    Torres-Juarez, Flor; Cardenas-Vargas, Albertina; Montoya-Rosales, Alejandra; González-Curiel, Irma; Garcia-Hernandez, Mariana H.; Enciso-Moreno, Jose A.; Hancock, Robert E. W.

    2015-01-01

    Tuberculosis is one of the most important infectious diseases worldwide. The susceptibility to this disease depends to a great extent on the innate immune response against mycobacteria. Host defense peptides (HDP) are one of the first barriers to counteract infection. Cathelicidin (LL-37) is an HDP that has many immunomodulatory effects besides its weak antimicrobial activity. Despite advances in the study of the innate immune response in tuberculosis, the immunological role of LL-37 during M. tuberculosis infection has not been clarified. Monocyte-derived macrophages were infected with M. tuberculosis strain H37Rv and then treated with 1, 5, or 15 μg/ml of exogenous LL-37 for 4, 8, and 24 h. Exogenous LL-37 decreased tumor necrosis factor alpha (TNF-α) and interleukin-17 (IL-17) while inducing anti-inflammatory IL-10 and transforming growth factor β (TGF-β) production. Interestingly, the decreased production of anti-inflammatory cytokines did not reduce antimycobacterial activity. These results are consistent with the concept that LL-37 can modulate the expression of cytokines during mycobacterial infection and this activity was independent of the P2X7 receptor. Thus, LL-37 modulates the response of macrophages during infection, controlling the expression of proinflammatory and anti-inflammatory cytokines. PMID:26351280

  12. Cough Aerosols of Mycobacterium tuberculosis Predict New Infection. A Household Contact Study

    PubMed Central

    Namugga, Olive; Mumbowa, Francis; Ssebidandi, Martin; Mbabazi, Olive; Moine, Stephanie; Mboowa, Gerald; Fox, Matthew P.; Reilly, Nancy; Ayakaka, Irene; Kim, Soyeon; Okwera, Alphonse; Joloba, Moses; Fennelly, Kevin P.

    2013-01-01

    Rationale: Airborne transmission of Mycobacterium tuberculosis results from incompletely characterized host, bacterial, and environmental factors. Sputum smear microscopy is associated with considerable variability in transmission. Objectives: To evaluate the use of cough-generated aerosols of M. tuberculosis to predict recent transmission. Methods: Patients with pulmonary tuberculosis (TB) underwent a standard evaluation and collection of cough aerosol cultures of M. tuberculosis. We assessed household contacts for new M. tuberculosis infection. We used multivariable logistic regression analysis with cluster adjustment to analyze predictors of new infection. Measurements and Main Results: From May 2009 to January 2011, we enrolled 96 sputum culture-positive index TB cases and their 442 contacts. Only 43 (45%) patients with TB yielded M. tuberculosis in aerosols. Contacts of patients with TB who produced high aerosols (≥10 CFU) were more likely to have a new infection compared with contacts from low-aerosol (1–9 CFU) and aerosol-negative cases (69%, 25%, and 30%, respectively; P = 0.009). A high-aerosol patient with TB was the only predictor of new M. tuberculosis infection in unadjusted (odds ratio, 5.18; 95% confidence interval, 1.52–17.61) and adjusted analyses (odds ratio, 4.81; 95% confidence interval, 1.20–19.23). Contacts of patients with TB with no aerosols versus low and high aerosols had differential tuberculin skin test and interferon-γ release assay responses. Conclusions: Cough aerosols of M. tuberculosis are produced by a minority of patients with TB but predict transmission better than sputum smear microscopy or culture. Cough aerosols may help identify the most infectious patients with TB and thus improve the cost-effectiveness of TB control programs. PMID:23306539

  13. Constitutive expression of SMAR1 confers susceptibility to Mycobacterium tuberculosis infection in a transgenic mouse model

    PubMed Central

    Yadav, Bhawna; Malonia, Sunil K.; Majumdar, Subeer S.; Gupta, Pushpa; Wadhwa, Neerja; Badhwar, Archana; Gupta, Umesh D.; Katoch, Vishwa M.; Chattopadhyay, Samit

    2015-01-01

    Background & objectives: Studies involving animal models of experimental tuberculosis have elucidated the predominant role of cytokines secreted by T cells and macrophages to be an essential component of the immune response against Mycobacterium tuberculosis infection. The immune activities of CD4+ T cells are mediated in part by Th1 cytokine interferon gamma (IFN-γ) which is produced primarily by T cells and natural killer (NK) cells and critical for initiating the immune response against intracellular pathogen such as M. tuberculosis. Nuclear matrix protein SMAR1 plays an important role in V(D)J recombination, T helper cell differentiation and inflammatory diseases. In this study a transgenic mouse model was used to study the role of SMAR1 in M. tuberculosis infection. Methods: Wild type BALB/c, C57BL/6, BALB/c-EGFP-SMAR1 and C57BL/6-SMAR1 transgenic mice were infected with M. tuberculosis (H37Rv). A dose of 100 bacilli was used for infection via respiratory route. Bacterial load in lung and spleen of infected mice was determined at 2, 4, 6 and 8 wk post-infection. Gene expression analysis for Th1 cytokines and inducible nitric oxide synthase (iNOS) was performed in infected lung tissues by quantitative reverse transcription (RT)-PCR. Results: SMAR1 transgenic mice from both BALB/c and C57BL/6 genetic background displayed higher bacillary load and susceptibility to M. tuberculosis infection compared to wild type mice. This susceptibility was attributed due to compromised of Th1 response exhibited by transgenic mice. Interpretation & conclusions: SMAR1 transgenic mice exhibited susceptibility to M. tuberculosis infection in vivo irrespective of genetic background. This susceptibility was attributed to downregulation of Th1 response and its hallmark cytokine IFN-γ. Hence, SMAR1 plays an important role in modulating host immune response after M. tuberculosis infection. PMID:26831422

  14. Mycobacterium tuberculosis Complex and HIV Co-Infection among Extrapulmonary Tuberculosis Suspected Cases at the University of Gondar Hospital, Northwestern Ethiopia

    PubMed Central

    Fanosie, Alemu; Gelaw, Baye; Tessema, Belay; Tesfay, Wogahta; Admasu, Aschalew; Yitayew, Gashaw

    2016-01-01

    Background Extrapulmonary Tuberculosis (EPTB) and Human Immunodeficiency Virus (HIV) infection are interrelated as a result of immune depression. The aim of this study was to determine the prevalence of Mycobacterium tuberculosis complex isolates and the burden of HIV co-infection among EPTB suspected patients. Method An institution based cross-sectional study was conducted among EPTB suspected patients at the University of Gondar Hospital. Socio-demographic characteristics and other clinical data were collected using a pretested questionnaire. GeneXpert MTB/RIF assay was performed to diagnosis Mycobacterium tuberculosis complex and Rifampicin resistance. All samples were also investigated by cytology and culture. The HIV statuses of all patients were screened initially by KHB, and all positive cases were further re-tested by STAT-pack. Data was analyzed using SPSS version 20 computer software and a P-value of < 0.05 was taken as statistically significant. Results A total of 141 extrapulmonary suspected patients were enrolled in this study. The overall prevalence of culture confirmed extrapulmonary tuberculosis infection was 29.8%, but the GeneXpert result showed a 26.2% prevalence of Mycobacterium tuberculosis complex infection. The 78.4% prevalence of extrapulmonary tuberculosis infection was found to be higher among the adult population. The prevalence of HIV infection among EPTB suspected patients was 14.1%, while it was 32.4% among GeneXpert-confirmed extrapulmonary TB cases (12/37). Tuberculosis lymphadenitis was the predominant (78.4%) type of EPTB infection followed by tuberculosis cold abscess (10.7%). Adult hood, previous history of contact with known pulmonary tuberculosis patients, and HIV co-infection showed a statistically significant association with extrapulmonary tuberculosis infection (P<0.013). Conclusion The prevalence of culture confirmed-EPTB infection was high, and a higher EPTB-HIV co-infection was also observed. PMID:26950547

  15. Heterogeneity of the repertoire of T cells of tuberculosis patients and healthy contacts to Mycobacterium tuberculosis antigens separated by high-resolution techniques.

    PubMed Central

    Schoel, B; Gulle, H; Kaufmann, S H

    1992-01-01

    In this report, we describe studies to examine the repertoire of freshly isolated human T lymphocytes to 400 distinct antigen fractions of Mycobacterium tuberculosis separated by a novel method involving two-dimensional gel electrophoresis. Separated antigens were probed with T cells from tuberculosis patients and purified protein derivative (PPD)-positive (PPD+) and PPD-negative (PPD-) contacts as well as normal healthy donors. T cells from all donors tested responded to separated antigens. Stimulation profiles for tuberculosis patients and PPD+ contacts were extremely heterogeneous, formally demonstrating that an enormous number of different antigens serve as targets of the cellular immune response to M. tuberculosis. Stimulation profiles for tuberculosis patients and PPD+ contacts were indistinguishable. However, stimulation profiles for tuberculosis patients and PPD+ contacts were easily distinguishable from those for PPD- contacts. Normal healthy donors showed T cell responses similar to those of either PPD+ or PPD- contacts. Images PMID:1548098

  16. Analysis of Immune Responses against a Wide Range of Mycobacterium tuberculosis Antigens in Patients with Active Pulmonary Tuberculosis

    PubMed Central

    Kassa, Desta; Ran, Leonie; Geberemeskel, Wudneh; Tebeje, Mekashaw; Alemu, Amelewerk; Selase, Alemayehu; Tegbaru, Belete; Franken, Kees L. M. C.; Friggen, Annemieke H.; van Meijgaarden, Krista E.; Ottenhoff, Tom H. M.; Wolday, Dawit; Messele, Tsehaynesh

    2012-01-01

    Characterizing host immune responses to molecular targets of Mycobacterium tuberculosis is essential to develop effective immunodiagnostics and better vaccines. We investigated the immune response against a large series of M. tuberculosis antigens, including 5 classical and 64 nonclassical (39 DosR regulon-encoded, 4 resuscitation-promoting factor [RPF], and 21 reactivation-associated) antigens in active-pulmonary-tuberculosis (TB) patients. Whole blood from TB patients (n = 34) was stimulated in vitro with M. tuberculosis antigens. Gamma interferon (IFN-γ) was measured after 7 days of stimulation, using an enzyme-linked immunosorbent assay (ELISA). The majority of the study participants responded to the classical M. tuberculosis antigens TB10.4 (84.8%), early secreted antigenic target-6 kDa (ESAT-6)/CFP-10 (70.6%), and purified protein derivative (PPD) (55.9%). However, only 26.5% and 24.2% responded to HSP65 and Ag85A/B, respectively. Of the 64 nonclassical antigens, 23 (33.3%) were immunogenic (IFN-γ levels, >62 pg/ml) and 8 were strong inducers of IFN-γ (IFN-γ levels, ≥100 pg/ml). The RPF antigens were the most immunogenic. In addition, we observed distinct cytokine expression profiles in response to several M. tuberculosis antigens by multiplex immunoassay. Tumor necrosis factor alpha (TNF-α), interleukin 10 (IL-10), and IL-6 were commonly detected at high levels after stimulation with 4/15 latency antigens (Rv0081, Rv2006, Rv2629, and Rv1733c) and were found especially in supernatants of the three strong IFN-γ inducers (Rv2629, Rv1009, and Rv2389c). IL-8, IL-6, and IL-17 were exclusively detected after stimulation with Rv0574c, Rv2630, Rv1998, Rv054c, and Rv2028c. In conclusion, in active-pulmonary-TB patients, we identified 23 new immunogenic M. tuberculosis antigens. The distinct expression levels of IFN-γ, TNF-α, IL-6, and IL-10 in response to specific subsets of M. tuberculosis antigens may be promising for the development of immunodiagnostics

  17. Simultaneous detection of Mycobacterium tuberculosis complex and nontuberculous mycobacteria in respiratory specimens.

    PubMed

    Hwang, Sang Mee; Lim, Mi Suk; Hong, Yun Ji; Kim, Taek Soo; Park, Kyoung Un; Song, Junghan; Lee, Jae Ho; Kim, Eui Chong

    2013-11-01

    Many nontuberculous mycobacteria (NTM) species have clinical significance, and the rapid and reliable identification of Mycobacterium tuberculosis complex (MTBC) and NTM species is important. We evaluated the simultaneous detection of MTBC and NTM in respiratory specimens. MTBC and NTM were simultaneously detected and identified by laboratory-developed (LDT) real-time PCR, multiplex real-time PCR/melting curve analysis, rpoB PCR restriction fragment length polymorphisms and the AdvanSure Mycobacteria GenoBlot assay (LG Life Sciences). Eighty-five respiratory specimens from 69 patients showed simultaneous detection of MTBC and NTM. A line probe assay showed 70.6% concordance with LDT. Ten patients (14.5%) had a history of tuberculosis, and eight patients (11.6%) had been previously diagnosed with bronchiectasis. Mixed cultures were present one time in 57 patients (82.6%) and repeatedly in 12 patients (17.4%). MTBC was more frequent in 44 patients (63.8%), and NTM was isolated in seven patients (10.1%). The commonly detected NTM species in the mixed cultures were Mycobacterium intracellulare (29.0%) and Mycobacterium abscessus (29.0%). Co-isolation caused a failure of antitubercular drug susceptibility testing in 2 patients (2.9%). Molecular methods allow MTBC and NTM species to be simultaneously identified in respiratory specimens. NTM isolated with MTBC has clinical significance in some patients and should not be ignored. PMID:23988279

  18. Simple and rapid method for detection of nitrate reductase activity of Mycobacterium tuberculosis and Mycobacterium canettii grown in the Bactec MGIT960 system.

    PubMed

    Goh, Khye Seng; Rastogi, Nalin

    2010-05-01

    Mycobacterium tuberculosis reduces nitrate very strongly as compared to Mycobacterium bovis and M. bovis BCG. Nitrate reductase, in conjunction with niacin accumulation, constitutes one of the major biochemical tests used in clinical microbiology laboratories to differentiate M. tuberculosis from other members of the M. tuberculosis complex, as well as nontuberculous Mycobacteria. Determination of nitrate reductase activity is currently performed using cultures grown on solid media with a slow detection time and the need for large quantities of bacilli, as otherwise the test is not reliable. Hereby, we propose a nitrate reduction test coupled to Bactec MGIT960 system as a simple, rapid and economic method with a total gain of time of about 3 to 4weeks over the conventional solid medium. In our study, almost all the M. tuberculosis and Mycobacterium canettii strains gave a strongly positive nitrate reductase result within 1day of positive detection by the MGIT960 system. In contrast, M. bovis, M. bovis BCG and M. africanum strains remained negative even after 14days of incubation. The possibility to detect nitrate reductase within 1 to 3days of a positive culture using MGIT960 opens new perspectives with the possibility of confirming M. tuberculosis - starting directly from pathological specimens. PMID:20298726

  19. Epidemiological Characterization of Drug Resistance among Mycobacterium tuberculosis Isolated from Patients in Northeast of Iran during 2012-2013

    PubMed Central

    Tavanaee Sani, Ashraf; Shakiba, Abolfazl; Bahrami Taghanaki, Hamid Reza; Ayati Fard, Seiedeh Fatemeh; Ghazvini, Kiarash

    2015-01-01

    Introduction. Tuberculosis is still one of the most important health problems in developing countries and increasing drug resistance is the main concern for its treatment. This study was designed to characterize the drug resistant Mycobacterium tuberculosis isolated from patients suffering from pulmonary tuberculosis in northeast of Iran. Method. In this cross-sectional study during 2012-2013, drug susceptibility testing was performed on Mycobacterium tuberculosis isolated in northeast of Iran using proportional method. Epidemiological data concerning these strains were also analyzed. Results. Among 125 studied isolates, 25 mycobacteria (20%) were diagnosed as nontuberculosis mycobacteria. Among the remaining 100 Mycobacterium tuberculosis isolates, the resistance rates were 7%, 7%, 3%, and 9% against isoniazid, rifampin, ethambutol, and streptomycin, respectively. Four isolates were resistant against both isoniazid and rifampin (MDR tuberculosis). The highest resistance rate was observed among 15–45-year-old patients. The MDR tuberculosis was much more prevalent among those who had previous history of treatment. Conclusion. Considering these findings, DOTS strategy should be emphasized and promptly used in order to prevent further resistance. Regarding the high rate of nontuberculosis mycobacteria, it is recommended that confirmatory tests were performed before any therapeutic decision. PMID:26064950

  20. IdeR is required for iron homeostasis and virulence in Mycobacterium tuberculosis

    PubMed Central

    Pandey, Ruchi; Rodriguez, G. Marcela

    2013-01-01

    Iron is an essential but potentially harmful nutrient, poorly soluble in aerobic conditions, and not-freely available in the human host. To acquire iron, bacteria have evolved high affinity iron acquisition systems that are expressed under iron limitation often in conjunction with virulence determinants. Because excess iron can be dangerous, intracellular iron must be tightly controlled. In mycobacteria, IdeR functions as a global iron dependent transcriptional regulator, but because inactivation of ideR is lethal for Mycobacterium tuberculosis, it has not been possible to use genetics to fully characterize this protein’s function or examine the requirement of iron regulation during tuberculosis infection. In this work, a conditional M. tuberculosis ideR mutant was generated and used to study the basis of IdeR’s essentiality. This investigation uncovered positive regulation of iron storage as a critical aspect of IdeR’s function in regular culture and a prominent factor for survival under stresses associated with life in macrophages. Moreover, this study demonstrates that IdeR is indispensable in the mouse model of tuberculosis, thereby linking iron homeostasis to virulence in M. tuberculosis. PMID:24205844

  1. Recognition of Stage-Specific Mycobacterial Antigens Differentiates between Acute and Latent Infections with Mycobacterium tuberculosis

    PubMed Central

    Demissie, Abebech; Leyten, Eliane M. S.; Abebe, Markos; Wassie, Liya; Aseffa, Abraham; Abate, Getahun; Fletcher, Helen; Owiafe, Patrick; Hill, Philip C.; Brookes, Roger; Rook, Graham; Zumla, Alimuddin; Arend, Sandra M.; Klein, Michel; Ottenhoff, Tom H. M.; Andersen, Peter; Doherty, T. Mark

    2006-01-01

    Mycobacterium tuberculosis is estimated to infect 80 to 100 million people annually, the majority of whom do not develop clinical tuberculosis (TB) but instead maintain the infection in a latent state. These individuals generally become positive in response to a tuberculin skin test and may develop clinical TB at a later date, particularly if their immune systems are compromised. Latently infected individuals are interesting for two reasons. First, they are an important reservoir of M. tuberculosis, which needs to be considered for TB control. Second, if detected prior to recrudescence of the disease, they represent a human population that is making a protective immune response to M. tuberculosis, which is very important for defining correlates of protective immunity. In this study, we show that while responsiveness to early secretory antigenic target 6 is a good marker for M. tuberculosis infection, a strong response to the 16-kDa Rv2031c antigen (HspX or α-crystallin) is largely restricted to latently infected individuals, offering the possibility of differential immunodiagnosis of, or therapeutic vaccination against, TB. PMID:16467323

  2. Evaluation of mtp40 genomic fragment amplification for specific detection of Mycobacterium tuberculosis in clinical specimens.

    PubMed Central

    Herrera, E A; Segovia, M

    1996-01-01

    A PCR assay based on the species-specific mtp40 genomic fragment was developed for the specific detection and identification of Mycobacterium tuberculosis in different uncultured clinical specimens. The aim of the study was to evaluate the clinical applicability of this target DNA in comparison with those of conventional microbiological methods and to compare the results obtained with those obtained after amplification with the IS6110 repetitive element. Discrepant results were interpreted in conjunction with the patients' clinical data, medical histories, and response to therapy. A total of 172 specimens from 162 patients with respiratory symptoms were tested, 101 specimens were obtained from 92 patients clinically suspected of having tuberculosis, and 71 specimens were obtained from 70 patients without known mycobacterial infection. The results of our study suggest that PCR amplification with the mtp40 genomic fragment provides a highly sensitive and specific technique for the detection of M. tuberculosis strains in clinical samples. It allows for the differentiation between M. tuberculosis and other related mycobacteria, including M. bovis, and is more specific than the IS6110 target. For these and other reasons, we propose that the mtp40 assay is a possible alternative for the specific direct detection of M. tuberculosis in clinical laboratories. PMID:8727885

  3. Critical role for NLRP3 in necrotic death triggered by Mycobacterium tuberculosis

    PubMed Central

    Wong, Ka-Wing; Jacobs Jr, William R

    2011-01-01

    Summary Induction of necrotic death in macrophages is a primary virulence determinant of Mycobacterium tuberculosis. The ESX-1 secretion system and its substrate ESAT-6 are required for M. tuberculosis to induce necrosis, but host factors that mediate the ESAT-6-promoted necrosis remain unknown. Here we report that ESAT-6-promoted necrotic death in THP-1 human macrophages is dependent on the NLRP3 inflammasome, as shown by RNA interference and pharmacological inhibitions. Phagosomes containing ESAT-6-expressing M. tuberculosis recruit markers previously associated with damaged phagosomal membrane, such as galectin-3 and ubiquitinated protein aggregates. In addition, ESAT-6 promoted lysosomal permeabilization by M. tuberculosis. ESAT-6 mutants defective for ubiquitination were unable to trigger NLRP3 activation and necrotic death. Furthermore, Syk tyrosine kinase, recently implicated in NLRP3 activation during fungal and malarial infections, was necessary for mediating the ESAT-6-promoted necrosis and NLRP3 activation. Our results thus link phagosomal damage and Syk activity to NLRP3-mediated necrotic death triggered by M. tuberculosis ESAT-6 during infection. PMID:21740493

  4. Restricted structural gene polymorphism in the Mycobacterium tuberculosis complex indicates evolutionarily recent global dissemination

    PubMed Central

    Sreevatsan, Srinand; Pan, Xi; Stockbauer, Kathryn E.; Connell, Nancy D.; Kreiswirth, Barry N.; Whittam, Thomas S.; Musser, James M.

    1997-01-01

    One-third of humans are infected with Mycobacterium tuberculosis, the causative agent of tuberculosis. Sequence analysis of two megabases in 26 structural genes or loci in strains recovered globally discovered a striking reduction of silent nucleotide substitutions compared with other human bacterial pathogens. The lack of neutral mutations in structural genes indicates that M. tuberculosis is evolutionarily young and has recently spread globally. Species diversity is largely caused by rapidly evolving insertion sequences, which means that mobile element movement is a fundamental process generating genomic variation in this pathogen. Three genetic groups of M. tuberculosis were identified based on two polymorphisms that occur at high frequency in the genes encoding catalase-peroxidase and the A subunit of gyrase. Group 1 organisms are evolutionarily old and allied with M. bovis, the cause of bovine tuberculosis. A subset of several distinct insertion sequence IS6110 subtypes of this genetic group have IS6110 integrated at the identical chromosomal insertion site, located between dnaA and dnaN in the region containing the origin of replication. Remarkably, study of ≈6,000 isolates from patients in Houston and the New York City area discovered that 47 of 48 relatively large case clusters were caused by genotypic group 1 and 2 but not group 3 organisms. The observation that the newly emergent group 3 organisms are associated with sporadic rather than clustered cases suggests that the pathogen is evolving toward a state of reduced transmissability or virulence. PMID:9275218

  5. CtpV: A putative copper exporter required for full virulence of Mycobacterium tuberculosis

    PubMed Central

    Ward, Sarah K.; Abomoelak, Bassam; Hoye, Elizabeth A.; Steinberg, Howard; Talaat, Adel M.

    2010-01-01

    Summary Copper is a required micronutrient that is also toxic at excess concentrations. Currently, little is known about the role of copper in interactions between bacterial pathogens and their human hosts. In this study, we elucidate a mechanism for copper homeostasis in the human pathogen Mycobacterium tuberculosis via characterization of a putative copper exporter, CtpV. CtpV was shown to be required by M. tuberculosis to maintain resistance to copper toxicity. Furthermore, the deletion of ctpV resulted in a 98-gene transcriptional response which elucidates the increased stress experienced by the bacteria in the absence of this detoxification mechanism. Interestingly, although the ΔctpV mutant survives close to the wild-type levels in both murine and guinea pig models of tuberculosis, animals infected with the ΔctpV mutant displayed decreased lung damage, and mutant-infected mice had a reduced immune response to the bacteria as well as a significant increase in survival time relative to mice infected with wild-type M. tuberculosis. Overall, our study provides the first evidence for a connection between bacterial copper response and the virulence of M. tuberculosis, supporting the hypothesis that copper response could be important to intracellular pathogens, in general. PMID:20624225

  6. Innate myeloid cell TNFR1 mediates first line defence against primary Mycobacterium tuberculosis infection.

    PubMed Central

    Segueni, Noria; Benmerzoug, Sulayman; Rose, Stéphanie; Gauthier, Amandine; Bourigault, Marie-Laure; Reverchon, Flora; Philippeau, Amandine; Erard, François; Le Bert, Marc; Bouscayrol, Hélène; Wachter, Thierry; Garcia, Irène; Kollias, George; Jacobs, Muazzam; Ryffel, Bernhard; Quesniaux, Valerie F.J.

    2016-01-01

    TNF is crucial for controlling Mycobacterium tuberculosis infection and understanding how will help immunomodulating the host response. Here we assessed the contribution of TNFR1 pathway from innate myeloid versus T cells. We first established the prominent role of TNFR1 in haematopoietic cells for controlling M. tuberculosis in TNFR1 KO chimera mice. Further, absence of TNFR1 specifically on myeloid cells (M-TNFR1 KO) recapitulated the uncontrolled M. tuberculosis